U.S. patent application number 12/118335 was filed with the patent office on 2008-10-30 for modified viral particles with immunogenic properties and reduced lipid content useful for treating and preventing infectious diseases.
This patent application is currently assigned to Lipid Sciences, Inc.. Invention is credited to Marc Bellotti, Bill E. Cham, Jo-Ann B. Maltais.
Application Number | 20080267997 12/118335 |
Document ID | / |
Family ID | 56290573 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080267997 |
Kind Code |
A1 |
Cham; Bill E. ; et
al. |
October 30, 2008 |
Modified Viral Particles with Immunogenic Properties and Reduced
Lipid Content Useful for Treating and Preventing Infectious
Diseases
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 viral
organisms are found in biological fluids, such as blood. The
present invention employs solvents useful for extracting lipids
from the lipid-containing infectious viral organism thereby
creating modified viral particles with reduced infectivity and
enhanced antigenicity. The present invention provides vaccine
compositions, comprising these modified viral particles with
reduced infectivity and enhanced antigenicity, optionally combined
with a pharmaceutically acceptable carrier or an immunostimulant.
The vaccine composition is administered to a patient to provide
protection against the lipid-containing infectious viral organism.
The vaccine compositions of the present invention include
combination vaccines of modified viral particles obtained from one
or more strains of a virus and/or one or more types of virus.
Inventors: |
Cham; Bill E.; (Port Vila,
VU) ; Maltais; Jo-Ann B.; (San Ramon, CA) ;
Bellotti; Marc; (Coto De Caza, 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: |
56290573 |
Appl. No.: |
12/118335 |
Filed: |
May 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10873015 |
Jun 21, 2004 |
7407662 |
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12118335 |
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10601656 |
Jun 20, 2003 |
7407663 |
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10873015 |
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10311679 |
Dec 18, 2002 |
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PCT/IB01/01099 |
Jun 21, 2001 |
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10601656 |
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PCT/AU00/01603 |
Dec 28, 2000 |
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10311679 |
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60390066 |
Jun 20, 2002 |
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60491928 |
Aug 1, 2003 |
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60533542 |
Dec 31, 2003 |
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60542947 |
Feb 9, 2004 |
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Current U.S.
Class: |
424/225.1 |
Current CPC
Class: |
A61M 1/3486 20140204;
C12N 7/00 20130101; A61P 37/00 20180101; C12N 2740/15034 20130101;
C12N 2730/10163 20130101; A61K 39/29 20130101; C12N 2740/15063
20130101; C12N 2730/10134 20130101; C12N 2770/24334 20130101; A61K
2039/5252 20130101; A61L 2/0088 20130101; A61K 39/292 20130101;
C12N 2770/24063 20130101; C12N 2770/20034 20130101; C12N 2770/24363
20130101; A61K 39/12 20130101; A61K 2039/55566 20130101; A61K
2039/57 20130101; A61L 2/0011 20130101 |
Class at
Publication: |
424/225.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/00 20060101 A61P037/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2000 |
AU |
PQ8469 |
Claims
1-69. (canceled)
70. A method of providing protection in a patient against an
infectious hepatitis viral particle comprising the step of:
administering to the patient an effective amount of a vaccine
composition comprising at least a partially delipidated hepatitis
viral particle having at least one exposed viral antigen that was
not exposed in a non-delipidated hepatitis viral particle, wherein
the amount is effective to provide a protective effect against
infection by the infectious hepatitis viral particle in the
patient.
71. The method of claim 70, further comprising administering a
pharmaceutically acceptable carrier or an immunostimulant.
72. The method of claim 70, wherein the protective effect comprises
enhanced interferon gamma production by T cells of the patient.
73. The method of claim 72, wherein the T-cells are CD4+ or CD8+
T-cells.
74. The method of claim 70, wherein the protective effect comprises
proliferation of cells of the immune system of the patient.
75. The method of claim 70, wherein the partially delipidated
hepatitis viral particle has a lower cholesterol content than the
non-delipidated hepatitis viral particle.
76. The method of claim 75, wherein the lower cholesterol content
is at least 20% lower than the non-delipidated viral particle.
77. The method of claim 75, wherein the lower cholesterol content
is at least 30% lower than the non-delipidated viral particle.
78. The method of claim 70, wherein the vaccine composition
comprises partially delipidated viral particles from one or more
strains of hepatitis virus or one or more types of hepatitis
virus.
79. The method of claim 70, wherein the partially delipidated
hepatitis viral particle has a different buoyant density than the
non-delipidated hepatitis viral particle.
80. A method for provoking a positive immune response in patient
having a plurality of lipid-containing hepatitis viral particles,
comprising the steps of: obtaining a fluid containing the
lipid-containing hepatitis viral particles from the patient;
contacting the fluid containing the lipid-containing hepatitis
viral particles with a first organic solvent capable of extracting
lipid from the lipid-containing hepatitis viral particles; mixing
the fluid and the first organic solvent; permitting organic and
aqueous phases to separate; collecting the aqueous phase containing
modified hepatitis viral particles with reduced lipid content; and
introducing the aqueous phase containing the modified hepatitis
viral particles with reduced lipid content into the patient wherein
the modified hepatitis viral particles with reduced lipid content
provoke an immune response in the patient.
81. The method of claim 80, further comprising: contacting the
aqueous phase with charcoal capable of removing the first organic
solvent from the aqueous phase.
82. The method of claim 80, wherein after the aqueous phase is
collected, the aqueous phase is contacted with charcoal capable of
removing the first organic solvent from the aqueous phase, and the
aqueous phase containing reduced levels of the first organic
solvent is eluted from the charcoal before introducing the aqueous
phase containing the modified hepatitis viral particles with
reduced lipid content into the patient.
83. The method of claim 80, wherein the first organic solvent is an
alcohol, an ether, an amine, a hydrocarbon, an ester, a surfactant,
or a combination thereof.
84. The method of claim 80, wherein the first organic solvent is an
alcohol, an ether, or a combination thereof.
85. The method of claim 80, wherein the ether is a C4 to C8 ether
and the alcohol is a C1 to C8 alcohol.
86. The method of claim 80, wherein the fluid is plasma, serum,
peritoneal fluid, lymphatic fluid, pleural fluid, pericardial
fluid, cerebrospinal fluid, or a fluid of the reproductive
system.
87. A method for treating a hepatitis viral infection in a patient
comprising: removing blood containing a plurality of
lipid-containing infectious hepatitis viral particles from the
animal or the human; obtaining plasma from the blood, the plasma
containing the lipid-containing infectious hepatitis viral
particles; contacting the plasma containing the lipid-containing
infectious hepatitis viral particles with a first organic solvent
capable of extracting lipid from the lipid-containing infectious
hepatitis viral particles to produce modified hepatitis 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
hepatitis viral particles; and introducing the aqueous phase
containing the modified hepatitis viral particles into the patient
wherein the modified hepatitis viral particles have at least one
exposed viral antigen that was not exposed in the plurality of
lipid-containing infectious hepatitis viral particles.
88. The method of claim 87, wherein after the aqueous phase is
collected, the aqueous phase is contacted with a charcoal capable
of removing the first organic solvent from the aqueous phase and
aqueous phase with reduced levels of first organic solvent is
eluted from the charcoal before introducing the aqueous phase
containing the modified hepatitis viral particles into the
patient.
89. The method of claim 87, wherein the first organic solvent is an
alcohol, an ether, an amine, a hydrocarbon, an ester, a surfactant
or a combination thereof.
90. The method of claim 87, wherein the first organic solvent is an
alcohol, an ether, or a combination thereof.
91. The method of claim 87, wherein the ether is a C4 to C8 ether
and the alcohol is a C1 to C8 alcohol.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
non-provisional patent application Ser. No. 10/601,656 filed Jun.
20, 2003, which is a continuation-in-part of U.S. non-provisional
patent application Ser. No. 10/311,679 filed Dec. 18, 2002, which
is a U.S. national phase from PCT patent application number
PCT/IB11/01099 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. U.S.
non-provisional patent application Ser. No. 10/311,679 claims the
benefit of U.S. provisional patent application Ser. No. 60/390,066
filed Jun. 20, 2002. The present application also claims the
benefit of U.S. provisional patent application Ser. No. 60/491,928
filed Aug. 1, 2003, 60/533,542 filed Dec. 31, 2003, and 60/542,947
filed Feb. 9, 2004.
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 cellular responses and antibody
production when introduced into a human or an animal. 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 virus. 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, T cells are activated on
recognizing the antigen displayed on the APC. There are two steps
in the cellular immune response. The first step involves activation
of cytotoxic T cells (CTL) or CD8.sup.+ T killer cells that
proliferate and kill target cells that specifically present
antigens. The second involves helper T cells (HTL) or CD4.sup.+ T
cells that regulate the production of antibodies and the activity
of CD8.sup.+ cells. The CD4.sup.+ T cells provide growth factors to
CD8.sup.+ 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". 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
toward 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 replication. 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. The most effective approach
to date for treating HIV is the use of highly active antiretroviral
therapy (HAART) which is expensive, toxic to the patient, and does
not eradicate the virus. Strict adherence to HAART regimen remains
a major hurdle, and lapses in compliance lead to bursts of viral
replication, and selection of drug resistant strains. Additionally,
long-term use of HAART is associated with side effects such as
lipodystrophies, altered glucose metabolism and elevated
cholesterol and triglycerides in plasma. There is, therefore, a
pressing need for additional therapies, either in form of
preventative and therapeutic vaccines, or development of
immunomodulating agents to augment HAART. The current approaches to
HIV vaccine development are reviewed by Mwau et al (2003. A review
of vaccines for HIV prevention. J Gene Med 5:3.). Briefly,
strategies include a variety of expression vectors, DNA based
recombinant vaccines, combinations of DNA based vaccines and viral
protein boosts with or without adjuvant. A recent Phase III
clinical trial using recombinant gp120 vaccine in Thailand, for
example, ended without success (Cohen, J. 2003. Public health. AIDS
vaccine still alive as booster after second failure in Thailand.
Science 302:1309), possibly because recombinant viral proteins need
to be in the correct configuration for appropriate immune responses
to be generated. Clearly, other novel approaches to enhancing
immune responses to viral antigens need to be evaluated.
[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 the
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. Viral inactivation does present
problems since 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. In sum, prior art
methods have largely focused on destroying, yet not suitably
modifying, viral particles to produce an immune response.
Current Methods of Manufacture of Viral Treatments and Medicaments
Viral Inactivation (or Chemical Kill)
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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
[0018] To date, several manufacturing methods have been employed in
search of 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 discrete 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.
[0019] 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.
[0020] 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 pathogens, via
heat or chemical means, into an individual introduces the pathogens
to the individual's immune system in a non-infective form thereby
initiating 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.
[0021] 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 basis 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.
[0022] 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 virus is
SHIV, which is a synthetic virus made from the HIV envelope and SIV
core.
[0023] What is needed is a therapeutic method and system for
providing patients with patient-specific viral antigens capable of
initiating a protective immune response. Accordingly, what is
needed is a simple, effective method that does not appreciably
denature or extract 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 further reduce viral infection and prevent further
infection.
[0024] 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 protective immune response with minimized risk of
infecting the individual.
SUMMARY OF THE INVENTION
[0025] 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 or non
host-derived modified viral particle that has its lipid envelope at
least partially removed, generating a positive immunologic response
when administered to a patient, thereby providing that patient with
some degree of protection against the virus. It is believed that
these modified viral particles have at least one antigen exposed
that was not exposed prior to the delipidation process.
[0026] 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, with reduced infectivity, and
such that an immune response is initiated upon reintroduction of
the fluid with reduced lipid content into the patient. This
autologous method ensures that patient specific antigens, for
example patient specific viral antigens, are introduced into the
same patient from which they were obtained to induce an immune
response. This is an important feature since a patient's physiology
may modify the antigens present in an infectious organism such as a
virus. 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 reduce the lipid content of the virus in
the plasma 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 with reduced lipid content is
administered to a patient, such as an animal or a human, optionally
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 or
separately.
[0027] The present method is also employed to produce
non-autologous vaccines, wherein biological fluids with lipid
containing viruses from at least one animal or human are treated to
produce a modified viral particle for administration into a
different (non-autologous) animal or human. The present invention
is also effective in producing an non-autologous, vaccine against
the virus, by treating a biological fluid such as plasma obtained
from an animal or a human with the present method to reduce lipid
levels in the fluid and in the virus within the fluid. Such treated
fluid with reduced lipid levels and containing modified virus with
reduced lipid levels may be introduced into another animal or human
which was not the source of the treated biological fluid. This
non-autologous method is employed to vaccinate a recipient animal
or human against one or more infectious organisms such as viruses.
Biological fluids may be used from animals or humans infected with
one or more infectious organisms such as viruses, and treated with
the present methods to produce a vaccine for administration to a
recipient animal or human. Alternatively, or in addition, various
stock supplies of virus may be added to a biological fluid before
treating the fluid with the method of the present invention to
create a vaccine.
[0028] The present invention encompasses vaccines made with the
delipidation method of the present invention that include more than
one strain of the same infectious organism, for example more than
one dade of HIV virus (e.g., HIV-1 and HIV-2). Such vaccines
provide an immune response to more than one strain of the same
infectious organism. Any number of different infectious strains or
clades of the same virus may be chosen and treated with the
delipidation method of the present invention to form numerous
vaccines. Alternatively, or in addition, various stock supplies of
different strains or clades of virus may be added to a biological
fluid before treating the fluid with the method of the present
invention to create a vaccine capable of generating an immune
response. Stocks of one or more viral preparation may be employed
to make a non-autologous vaccine directed to one or more viruses.
In this manner combination vaccines are produced which provide
protection against multiple strains or clades of a virus or against
multiple viruses.
[0029] The present invention encompasses vaccines made with the
delipidation method of the present invention that include more than
one infectious organism, such as more than one virus. Such
combination vaccines provide an immune response to more than one
infectious organism, for example, HIV and hepatitis. Any number of
different infectious organisms may be chosen and treated with the
delipidation method of the present invention to form numerous
combination vaccines.
[0030] Thus an effective method is presented, by which new vaccines
can be developed from 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 an immune response when re-introduced into the
patient.
[0031] The present invention provides a modified viral particle
comprising at least a partially delipidated viral particle, wherein
the partially delipidated viral particle initiates an immune
response in a patient and incites protection against an infectious
organism in the patient.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] The present invention also provides a vaccine composition,
comprising at least a partially delipidated viral particle having
patient-specific viral antigens and optionally 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.
[0036] 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.
[0037] The present invention also provides a method to protect a
patient against an infectious viral particle comprising
administering to the patient 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 optionally 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.
[0038] The present invention also provides a method for provoking a
positive immune response in a patient having a plurality of
lipid-containing viral particles, comprising the steps of:
obtaining a fluid containing the lipid-containing viral particles
from the patient; 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.
[0039] The present invention also provides a method for treating a
viral infection in a patient comprising: removing blood containing
a plurality of lipid-containing infectious viral particles from the
patient; 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; removing residual solvent from the aqueous phase;
and, introducing the aqueous phase containing the modified viral
particles into the patient 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. Introduction of these modified viral particles into the
patient produces an immune response to treat or lessen the severity
of the viral infection.
[0040] The present invention also provides a method for treating a
viral infection in a patient comprising: obtaining a fluid
comprising plurality of lipid-containing infectious viral particles
from a plurality of patients; optionally combining the
lipid-containing infectious viral particles with a suitable
biologically acceptable carrier; contacting the fluid containing
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 carrier 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 a different patient wherein the
modified viral particles have at least one exposed antigen that was
not exposed in the plurality of lipid-containing infectious viral
particles. In this embodiment, the lipid-containing infectious
viral particles represent one or more viral strains or one or more
types of virus and are not patient specific. Introduction of these
modified viral particles into the patient produces an immune
response to treat or lessen the severity of the viral
infection.
[0041] 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.
[0042] 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.
[0043] 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 and clades 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. Another preferred
virus treated with the method of the present invention is the
coronavirus SARS. 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.
[0044] 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.
[0045] It is an object of the present invention to provide a method
for treating lipid containing virus in order to create modified
viral particles with reduced lipid content while substantially
unaffecting protein levels when compared to unmodified viral
particles.
[0046] Yet another object of the present invention is to provide a
method for treating lipid containing virus in order to create
modified viral particles with reduced lipid content, with
substantially unaffected protein levels when compared to unmodified
viral particles, and with at least one exposed antigen associated
with the viral particles that was substantially unexposed in
unmodified viral particles.
[0047] It is another object of the present invention to provide a
method for treating or preventing viral disease by administering to
a patient modified viral particles with reduced lipid content and
at least one exposed antigen associated with the viral particles
that was substantially unexposed in unmodified viral particles.
[0048] 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.
[0049] Yet another object of the present invention is to provide a
method for creating, in a biological fluid, a plurality of modified
lipid containing viral particles having a distribution of reduced
lipid content, with a substantial percentage of viral particles
having substantially unaffected protein levels when compared to
unmodified viral particles.
[0050] 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.
[0051] 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 viral antigens.
[0052] 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.
[0053] Another object of the present invention is to completely or
partially delipidate viral particles, wherein the viral particles
comprise immunodeficiency virus, hepatitis in its various forms,
coronavirus, or any other lipid-containing virus, thereby creating
a modified viral particle.
[0054] It is a further object of the present invention to
completely or partially delipidate viral particles, wherein the
viral particles comprise immunodeficiency virus, hepatitis in its
various forms, coronavirus, or any other lipid-containing virus,
while retaining the structural protein core of the virus.
[0055] 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.
[0056] 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 modified viral particle with reduced lipid content which may be
administered to an animal or a human, optionally 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.
[0057] Still 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 modified viral particle with reduced lipid content which may be
administered to an animal or a human optionally with a
pharmaceutically acceptable carrier and optionally an
immunostimulant compound, to initiate a positive immunogenic
response in the animal or human.
[0058] It is another specific object of the present invention to
provide an anti-viral vaccine.
[0059] Another specific object of the present invention is to
provide an anti-viral vaccine that induces cellular responses in
cells of the immune system, wherein the cellular responses include
but are not limited to proliferation of cells and production of
immune system molecules such as interferon gamma.
[0060] 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, optionally combined with a pharmaceutically acceptable
carrier.
[0061] It is another object of the present invention to combine
viral particles with reduced lipid content having patient specific
antigens with delipidated stock viral particles with reduced lipid
content to create a therapeutic combination vaccine for the
treatment or prevention of more than one viral disease.
[0062] 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
[0063] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate preferred embodiments
of the present invention.
[0064] FIG. 1 depicts the density of sucrose gradient fractions as
indicated by the graphing of density against fraction number for
HIV viral particles subjected to delipidation using 1% DIPE, 1%
butanol/DIPE, 1% butanol, 2% butanol, and 5% butanol, along with a
control group.
[0065] FIG. 2 depicts the p2.sup.4 protein concentration (ng/ml)
for each of the fraction numbers shown in FIG. 1.
[0066] FIG. 3 is similar to FIG. 2 and is a schematic
representation of an isopycnic gradient analysis of delipidated HIV
subjected to delipidation using 1% DIPE, 1% butanol/DIPE, 1%
butanol, 2% butanol, and 5% butanol, along with a control group,
indicated by a graphing p24 levels as a percent of total recovered
p24 protein against fraction number.
[0067] FIG. 4 is a schematic representation of an isopycnic
gradient analysis of delipidated SIV-mac 251, indicated by a
graphic of gag p27 concentration (ng/ml) against fraction number
following delipidation conditions 1% DIPE, 5% DIPE:n-butanol
(75:25) and 1% n-butanol.
[0068] FIG. 5 is a schematic representation of a fast performance
liquid chromatography (FPLC) of the control and 1% DIPE-treated SIV
mac 521 showing the p27 gag levels (ug/ml) in each fraction
number.
[0069] FIG. 6 presents cholesterol levels (ng/ml) in the fractions
shown in FIG. 5.
[0070] FIG. 7 is a schematic representation of SIV mac 521
infectivity (TCID 50/ml) versus viral RNA copy numbers (copies/mg)
after 1% DIPE treatment, in live virus, and after AT-2
treatment.
[0071] FIGS. 8A and 8B show CD4.sup.+ and CD8.sup.+ T cell
responses (% interferon gamma positive cells) to SIV env (8A)
peptide pools and to SIV gag (8B) peptide pools in 1 million PMBCs
from AT-2 inactivated SIV primed mice boosted with live virus, AT-2
inactivated virus or delipidated virus (1% DIPE). Mean of 6 mice+or
-SEM are shown. **=p value <0.01, *=p value <0.05.
[0072] FIG. 9 is a schematic representation of SIV env gp120
antibody titers (O.D. at 450 nm) in mice immunized with AT-2
treated virus (SIV mac 251) and boosted with 1 ug total viral
protein of live virus (SIV mac 251), AT-2 inactivated virus or
delipidated virus (1% DIPE). Serial dilution of mouse plasma was
measured in ELISA plates coated with recombinant SIV mac251 gp120
env protein.
[0073] FIG. 10 is a schematic representation of SIV gag p55
antibody titers (O.D. at 450 nm) in mice immunized with AT-2
treated virus and boosted with 1 ug total viral protein of live
virus (SIV mac 251), AT-2 inactivated virus or delipidated virus
(1% DIPE). Serial dilution of mouse plasma was measured in ELISA
plates coated with recombinant SIV mac251 p55 gag protein.
[0074] FIG. 11 is a schematic representation of a correlation curve
of CD4.sup.+ responses (% IFN gamma cells) to SIV mac 251 Gag and
Env peptide pools to the antibody responses (O.D. 450 nm) to
recombinant Gag and Env. A strong correlation (R2=0.9993) was
observed between the cellular responses (CD4) to SIV mac 251 gag
and the anti-gag antibody responses. A good correlation (R2=0.953)
was observed between cellular responses (CD4.sup.+) to SIV mac 251
env and the anti-env antibody responses.
[0075] FIG. 12 presents the percentage of CD4.sup.+ cells
immunoreactive for IFN gamma in response to gag or env peptide
pools in four monkeys, each primed with an equivalent of 5 ug p24
HIV-IIIB in incomplete Freund's Adjuvant, and later boosted with 1
ug DIPE delipidated HIV-IIIB every month (RIl & RFo), or with 1
ug live HIV-IIIB every month (RFt & Rom).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0076] 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 blood, 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.
[0077] 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 in the fluid. 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.
[0078] The term "second extraction solvent" is defined as one or
more solvents that may be employed to 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.
[0079] 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.
[0080] 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.
[0081] The term "patient" refers to animals and humans.
[0082] The term "patient specific antigen" refers to an antigen
that is capable of inducing a patient specific immune response when
introduced into that patient. Such patient specific antigens may be
viral antigens. A patient specific antigen includes any antigen,
for example a viral antigen, that has been modified or influenced
within the patient.
A Modified Viral Particle
[0083] 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 have lower levels of cholesterol and
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. A schematic
representation of HIV viral particles contain the lipid containing
envelope or bilayer derived from a host cell, surface
glycoproteins, transmembrane proteins, the capsid, capsid proteins
and nuclear material is presented on page 238 of Robbins Pathologic
Basis of Disease (Cotran et al. eds 6.sup.th edition, W. B.
Saunders Co., 1999). 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, reduced infectivity and is immunogenic.
Modified Viral Particle Resulting from Removal of Lipid from
Lipid-Containing Organisms
[0084] Methods of the present invention solve numerous problems
encountered with prior art methods. By substantially 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 recognizes the viral particle as 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.
[0085] 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 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. Non-autologous vaccines are also created in the
present invention which are administered to patients that are
different from the source of the virus to be delipidated.
Combination vaccines directed against multiple viruses are also
within the scope of the present invention. Such combination
vaccines may be made from various biological fluids, from stock
supplies of multiple viruses (e.g., HIV, hepatitis and SARS) and/or
from multiple strains or clades of a virus (e.g., HIV-1 and
HIV-2).
Infectious Organisms Treated with the Present Invention
[0086] 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-Barr-like viruses), Rhadinovirus (saimiri-ateles-like
herpes viruses), Orthopoxvirus (orthopoxviruses), Parapoxvirus
(parapoxviruses), Avipoxvirus (fowlpox viruses), Capripoxvirus
(sheeppox-like 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, Poxyiridae, and any
other lipid-containing virus.
[0087] 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, sand fly 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, Arenavirus: 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
[0088] 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 with 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
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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; ketones; 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.
[0093] 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.
[0094] 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. Charcoal is a preferred method of
removing solvents. Pervaporation may also be employed to remove one
or more solvents from delipidated viral mixtures.
[0095] 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.
[0096] 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.
[0097] 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). Low concentrations of ethers may also be used in combination
with alcohols, for example, n-butanol.
[0098] 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.
[0099] 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.
[0100] 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 range from about 0.01 parts
alcohol to 99.99 parts ether to 60 parts alcohol to 40 parts ether,
with a specific ratio range of about 10 parts alcohol to 90 parts
ether to 5 parts alcohol to 95 parts ether, with a specific ratio
range of about 10 parts alcohol to 90 parts ether to 50 parts
alcohol to 50 parts ether, with a specific ratio range of about 20
parts alcohol to 80 parts ether to 45 parts alcohol to 55 parts
ether, with a specific range of about 25 parts alcohol to 75 parts
ether.
[0101] 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 parts butanol to 99.99 parts DIPE to 60 parts butanol to
40 parts DIPE, with a specific ratio range of about 10 parts
butanol to 90 parts DIPE to 5 parts butanol to 95 parts DIPE, with
a specific ratio range of about 10 parts butanol to 90 parts DIPE
to 50 parts butanol to 50 parts DIPE, with a specific ratio range
of about 20 parts butanol to 80 parts DIPE to 45 parts butanol to
55 parts DIPE, with a specific range of about 25 parts butanol to
75 parts DIPE.
[0102] 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 parts butanol to 99.99 parts DEE to 60 parts butanol
to 40 parts DEE, with a specific ratio range of about 10 parts
butanol to 90 parts DEE to 5 parts butanol to 95 parts DEE with a
specific ratio range of about 10 parts butanol to 90 parts DEE to
50 parts butanol to 50 parts DEE, with a specific ratio range of
about 20 parts butanol to 80 parts DEE to 45 parts butanol to 55
parts DEE, with a specific range of about 40 parts butanol to 60
parts 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
[0103] 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. 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.
[0104] Viruses in the plasma are affected by the treatment of the
plasma with the method of the present invention. The
lipid-containing viral organism may be separated from the red and
white cells using techniques known to one of ordinary skill in the
art.
[0105] Biological fluids include stocks of viral preparations
including various strains of viruses as well as different types of
viruses. Treatment of such biological fluids with the method of the
present invention produces modified viral particles that may be
administered to a patient as a non-autologous vaccine. Such
non-autologous vaccines provide protection in the patient against
more than strain of a virus and/or against more than one type of
virus. Treatment of lipid-containing organisms may occur in
biological fluids other than blood and plasma. For example,
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 non-blood types of fluids
affects the lipid-containing organisms in the fluid, such as the
virus.
[0106] 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.
[0107] 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.
[0108] 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
[0109] 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.
[0110] 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.
[0111] A preferred method of removing solvent is through the use 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.
[0112] 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. A preferred 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. In yet
another embodiment, 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.
[0113] 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.
Methods of Treating Biological Fluids (Delipidation)
[0114] 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.
[0115] 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, charcoal, pervaporation or 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.
[0116] 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,
the fluid with reduced lipid levels and containing virus with
reduced lipid levels 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 infectivity of the
infectious organism contained within the vascular system of the
human or animal. The modified viral particle also serves to
initiate an autologous immune response in the patient when
administered to the patient. 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.
[0117] 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 fluid may be contained within a
blood bank or in the alternative, drawn from a human or animal
prior to application of the method. The fluid may be a reproductive
fluid or any fluid used in the process of artificial insemination
or in vitro fertilization. The fluid 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. Stocks
of various strains or clades of a virus and also stocks of multiple
viruses may be used in the present method to produce vaccines. In
this mode of operation, this fluid is treated with the method of
the present invention to produce a new fluid with reduced lipid
levels 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 other animals or humans and stored in a
blood bank for subsequent transfusion. This is a non-autologous
method of providing vaccine protection. These samples may be
treated with the method of the present invention to treat or
prevent one or more infectious disease, such as HIV, hepatitis,
and/or cytomegalovirus, from the biological sample.
[0118] 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.
[0119] Through the use of the 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 with reduced infectivity,
act as a vaccine and provide protection in the patient against the
virus or provide a treatment in an infected patient by generating
an immune response and decreasing the severity of the disease.
These modified viral particles induce an immune response in the
recipient to exposed epitopes on the modified viral particles.
Alternatively the modified viral particles may be 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
[0120] In one embodiment, the modified viral particle, which is at
least partially or substantially delipidated and has immunogenic
properties, is optionally combined with a pharmaceutically
acceptable carrier to make a composition comprising a vaccine. In a
preferred embodiment, the modified viral particle is retained in
the biological fluid, such as plasma, with reduced lipid levels and
is administered to a patient as a vaccine. This vaccine composition
is optionally combined with an adjuvant or an immunostimulant and
administered to an animal or a human. Both autologous and
non-autologous vaccines, including combination vaccines, are within
the scope of the present invention. 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 strain of a virus or more than one viral
disease after vaccination. Such combinations may be selected
according to the desired immunity. For example, preferred
combinations include, 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. The
remaining modified viral particles of the organism are retained in
the delipidated biological fluid, and when reintroduced into the
animal or human, are presumably ingested by phagocytes and generate
an immune response.
Administration of Vaccine Produced With the Method of the Present
Invention
[0121] 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
optionally 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.
[0122] The formulations may be presented in unit-dose or multi-dose
containers--for example, sealed ampules and vials--and may be
stored in 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.
[0123] 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.
[0124] 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. The vaccine may also be
administered in the vicinity of lymphatic tissue, for example
through administration to the lymph nodes such as axillary,
inguinal or cervical lymph nodes.
[0125] 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 a patient.
Initial injections may range from about less than 1 ng to 1 gram
based on total viral protein. A non-limiting range may be 1 ml to
10 ml. The volume of administration may vary depending on the
administration route.
Vaccination Schedule
[0126] 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 time for
administration of the vaccine before initial infection is known to
one of ordinary skill in the art. However, the vaccine may also be
administered after initial infection to ameliorate disease
progression or to treat the disease.
Adjuvants
[0127] 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
[0128] 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.
[0129] 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
A. Delipidation of Serum Produces Duck Hepatitis B virus (DHBV)
Having Reduced Infectivity
[0130] 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.
[0131] The organic solvent system was mixed in the ratio of 40
parts butanol to 60 parts 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.
[0132] The positive and negative control duck sera were diluted in
phosphate buffered saline (PBS). Positive controls: 2 ml of pooled
serum containing 10.sup.6 ID.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.
[0133] 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.
[0134] 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
[0135] 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 positive following kill)]
challenge. GROUP 3 6 Mock Vaccine 6/6 ducks became DHBV (Control)
[Phosphate Buffered Saline positive following (PBS)] challenge.
1. Glutaraldehyde Inactivation
[0136] 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.
2. Delipidation Procedure
[0137] An organic solvent system was employed to perform
delipidation of serum. The solvent system consisted 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.
3. Vaccine Production
TABLE-US-00002 [0138] TABLE 2 Second Dose First Dose (injected with
300 .mu.l Third Dose (injected with 200 .mu.l of of respective
(injected with 300 .mu.l of respective vaccine into 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 A 200 .mu.l aliquot of the delipidated serum was the
delipidated delipidated serum was mixed mixed with 1960 .mu.l of
serum was mixed with 1800 .mu.l of PBS and then phosphate buffered
saline with 1960 .mu.l of PBS emulsified in 1000 .mu.l of Freund
(PBS) and then emulsified Incomplete Adjuvant. in 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 (DHBV positive
serum pool #4 DHBV positive serum positive serum pool #4 (20.4.99
SERUM (20.4.99) was mixed with pool #4 (20.4.99) was was mixed with
300 .mu.l of PBS CONTROL) 300 .mu.l of PBS and 100 .mu.l of a mixed
with 300 .mu.l of and 100 .mu.l 2% glutaraldehyde solution PBS and
100 .mu.l Aidal Plus (Whiteley Chemical (Aidal Plus from Whiteley
Aidal Plus (Whiteley and incubated for 10 minutes to Chemicals) and
incubated Chemicals) and inactivate the DHBV. A 40 .mu.l for 10
minutes to inactivate incubated aliquot of the inactivated the
DHBV. A 40 .mu.l aliquot for 10 minutes to serum/PBS mixture was
added of the inactivated inactivate the DHBV. 1960 .mu.l PBS and
emulsified in serum/PBS mixture was A 40 .mu.l aliquot of the 1000
.mu.l Freunds Incomplete added to 1960 .mu.l 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.
4. Experimental Procedure
[0139] Ducks were challenged with 100011 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).
5. Analysis of Results
[0140] a. Test ducks. 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.
[0141] b. Sham vaccinated ducks. All 4 of the ducks vaccinated with
glutaraldehyde inactivated serum became DHBV positive following
challenge with DHBV.
[0142] c. Mock vaccinated ducks. All 6 of the 6 mock-vaccinated
negative control ducks became DHBV positive following
challenge.
[0143] 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).
[0144] 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 6 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 Model for Hepatitis C
[0145] 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.
[0146] 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 106.8 infectious viral particles per ml of
clarified (centrifuged) culture medium.
1. Treating Infectious BVDV
[0147] 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.
[0148] 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.
2. Testing of treated BVDV Preparation a Tissue-Culture
Inoculation
[0149] Two milliliters 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).
[0150] 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.
[0151] b. Animal Inoculation
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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
[0157] 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).
[0158] 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
Use of Delipidated SIV to Induce or Augment SIV Specific Humoral
and CD4.sup.+ T Cell Memory Responses in Mice--a Model for a New
Auto-vaccination Strategy Against Lentiviral Infection
[0159] The following studies focused on the simian equivalent of
human HIV, termed SIV. The purpose was to utilize delipidated-SIV
mac251 (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 SIVmacl42 (96%). (Regier D A, Desrosiers Annual Review
Immunology. 1990; 8:557-78.)
[0160] 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.
[0161] 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, Ill.). 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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 106.7
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.
[0166] 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
[0167] 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.
[0168] 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.
[0169] 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.
[0170] 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.
[0171] 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
[0172] 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 10 ug dil. No boost 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
[0173] 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.sup.+ and CD8.sup.+ T cells to synthesize
IFN-gamma by standard intracellular cytokine staining (ICC) and
flow cytometry.
[0174] 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 SIV envelope (SE) peptides,
ranging from 8 to 9 peptides per pool depending on the pool (n=17
pools), or b) 0.1 ml of medium containing 2 ug/ml of each of a pool
of SIV gag (SG) peptides, ranging from 7 to 8 peptides per pool
depending on the pool (n=16 pools). 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 SIV env peptides (n=72 individual
peptides) were mixed in a grid fashion of an 8.times.9 matrix and
the SIV gag peptides (n=62 peptides with two pools missing a
peptide each and one pool missing two peptides) were mixed in a
grid fashion of an 8.times.8 matrix which permitted identification
of individual peptide specific immune responses. The SIV gag
peptides were generally synthetic 20 mer peptides that overlapped
each other by 12 amino acids and encompassed the entire SIV gag
sequence. The SIV env peptides were generally synthetic 25 mer
peptides that overlapped each other by 13 amino acids and
encompassed the entire SIV env 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 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 SIV mac 239
gag peptides (20-mers) overlap by 12 Pool Pool Pool Pool Pool 1
Pool 2 3 Pool 4 5 6 Pool 7 8 Pool 9 Sg 1 Sg 2 Sg 3 Sg 4 Sg 5 Sg 6
Sg 7 Sg 8 Pool 10 Sg 9 Sg 10 Sg 11 Sg 12 Sg 13 Sg 14 Sg 15 Sg 16
Pool 11 Sg 17 Sg 18 Sg 19 Sg 20 Sg 21 Sg 22 Sg 23 Sg 24 Pool 12 Sg
25 Sg 26 Sg 27 Sg 28 Sg 29 Sg 30 Sg 31 Sg 32 Pool 13 Sg 33 Sg 34 Sg
35 Sg 36 Sg 37 Sg 38 Sg 39 Sg 40 Pool 14 Sg 41 Sg 42 Sg 43 Sg 44 Sg
45 Sg 46 Sg 47 Sg 48 Pool 15 Sg 49 Sg 50 Sg 51 Sg 52 Sg 53 Sg 54 Sg
55 Sg 56 Pool 16 Sg 57 Sg 58 Sg 59 Sg 60 Sg 61 Sg 62
TABLE-US-00006 TABLE 6 Pool arrangement of individual SIV mac239
env peptides (25-mer) overlapping by 13 Pool Pool Pool Pool Pool 1
Pool 2 3 Pool 4 5 Pool 6 7 8 Pool 9 Se 1 Se 2 Se 3 Se 4 Se 5 Se 6
Se 7 Se 8 Pool 10 Se 9 Se 10 Se 11 Se 12 Se 13 Se 14 Se 15 Se 16
Pool 11 Se 17 Se 18 Se 19 Se 20 Se 21 Se 22 Se 23 Se 24 Pool 12 Se
25 Se 26 Se 27 Se 28 Se 29 Se 30 Se 31 Se 32 Pool 13 Se 33 Se 34 Se
35 Se 36 Se 37 Se 38 Se 39 Se 40 Pool 14 Se 41 Se 42 Se 43 Se 44 Se
45 Se 46 Se 47 Se 48 Pool 15 Se 49 Se 50 Se 51 Se 52 Se 53 Se 54 Se
55 Se 56 Pool 16 Se 57 Se 58 Se 59 Se 60 Se 61 Se 62 Se 63 Se 64
Pool 17 Se 65 Se 66 Se 67 Se 68 Se 69 Se 70 Se 71 Se 72
TABLE-US-00007 TABLE 7 SIV mac 239 gag peptides. These peptides are
generally 20 mers overlapping by 12 amino acids. They were selected
for synthesis, with the proviso that there was no Q at the amino
terminus, and no P in last or second to last position at the
carboxy terminus). SEQ ID NO:1 MGVRNSVLSGKKADELEKIR SG 1 1-20 SEQ
ID NO:2 SGKKADELEKIRLRPNGKKK SG 2 9-28 SEQ ID NO:3
EKIRLRPNGKKKYMLKHVVW SG 3 17-36 SEQ ID NO:4 GKKKYMLKHVVWAANELDRF SG
4 25-44 SEQ ID NO:5 HVVWAANELDRFGLAESLLE SG 5 33-52 SEQ ID NO:6
LDRFGLAESLLENKEGCQKI SG 6 41-60 SEQ ID NO:7 SLLENKEGCQKILSVLAPLV SG
7 49-68 SEQ ID NO:8 CQKILSVLAPLVPTGSENLK SG 8 57-76 SEQ ID NO:9
APLVPTGSENLKSLYNTVCV SG 9 65-84 SEQ ID NO:10 ENLKSLYNTVCVIWCIHAEE
SG 10 73-92 SEQ ID NO:11 TVCVIWCIHAEEKVKHTEEA SG 11 81-100 SEQ ID
NO:12 HAEEKVKHTEEAKQIVQRHL SG 12 89-108 SEQ ID NO:13
TEEAKQIVQRHLVVETGTT SG 13 97-115 SEQ ID NO:14 VQRHLVVETGTTETMPKTSR
SG 14 104-123 SEQ ID NO:15 GTTETMPKTSRPTAPSSGRG SG 15 113-132 SEQ
ID NO:16 TSRPTAPSSGRGGNYPVQQI SG 16 121-140 SEQ ID NO:17
SGRGGNYPVQQIGGNYVHL SG 17 129-147 SEQ ID NO:18 PVQQIGGNYVHLPLSPRTLN
SG 18 136-155 SEQ ID NO:19 YVHLPLSPRTLNAWVKLIEE SG 19 144-163 SEQ
ID NO:20 RTLNAWVKLIEEKKFGAEVV SG 20 152-171 SEQ ID NO:21
LIEEKKFGAEVVPQFQALSE SG 21 160-179 SEQ ID NO:22
AEVVPGFQALSEGCTPYDIN SG 22 168-187 SEQ ID NO:23
ALSEGCTPYDINQMLNCVQD * SG 23 176-195 SEQ ID NO:24
YDINQMLNCVGDHQAAMQII SG 24 184-203 SEQ ID NO:25
CVGDHQAAMQIIRDIINEEA SG 25 192-211 SEQ ID NO:26 MQIIRDIINEEAADWDLQH
SG 26 200-218 SEQ ID NO:27 NEEAADWDLQHPQPAPQQGQ SG 27 208-227 SEQ
ID NO:28 LQHPQPAPQQGQLREPSGSDI SG 28 216-236 SEQ ID NO:29
GQLREPSGSDIAGTTSSVDE SG 29 226-245 SEQ ID NO:30
SDIAGTTSSVDEQIQWMYRQ SG 30 234-253 SEQ ID NO:31
SVDEQIQWMYRQQNPIPVGN SG 31 242-261 SEQ ID NO:32
MYRQQNPIPVGNIYRRWIQL SG 32 250-269 SEQ ID NO:33
PVGNIYRRWIQLGLQKCVRM SG 33 258-277 SEQ ID NO:34
WIQLGLQKCVRMYNPTNILD SG 34 266-285 SEQ ID NO:35 CVRMYNPTNILDVKQGPKE
SG 35 274-292 SEQ ID NO:36 TNILDVKQGPKEPFQSYVDR SG 36 281-300 SEQ
ID NO:37 GPKEPFQSYVDRFYKSLRAE SG 37 289-308 SEQ ID NO:38
YVDRFYKSLRAEQTDAAVKN SG 38 297-316 SEQ ID NO:39
LRAEQTDAAVKNWMTQTLLI SG 39 305-324 SEQ ID NO:40
AVKNWMTQTLLIQNANPDCK SG 40 313-332 SEQ ID NO:41
TLLIQNANPDCKLVLKGLGV SG 41 321-340 SEQ ID NO:42
PDCKLVLKGLGVNPTLEEML SG 42 329-348 SEQ ID NO:43
GLGVNPTLEEMLTACQGVGG SG 43 337-356 SEQ ID NO:44
EEMLTACQGVGGPGQKARLM SG 44 345-364 SEQ ID NO:45
GVGGPGQKARLMAEALKEAL SG 45 353-372 SEQ ID NO:46
ARLMAEALKEALAPVPIPFA SG 46 361-380 SEQ ID NO:47
KEALAPVPIPFAAAQQRGPRK SG 47 369-389 SEQ ID NO:48
PFAAAQQRGPRKPIKCWNCG SG 48 378-397 SEQ ID NO:49
GPRKPIKCWNCGKEGHSARQ SG 49 386-405 SEQ ID NO:50
WNCGKEGHSARQCRAPRRQG SG 50 394-413 SEQ ID NO:51
SARQCRAPRRQGCWKCGKMD SG 51 402-421 SEQ ID NO:52
RRQGCWKCGKMDHVMAKCPTA SG 52 410-430 SEQ ID NO:53
KMDHVMAKCPDRQAGFLGLG SG 53 419-438 SEQ ID NO:54
CPDRQAQFLGLGPWGKKPRN SG 54 427-446 SEQ ID NO:55
LGLGPWGKKPRNFPMAQVHQ SG 55 435-454 SEQ ID NO:56 KPRNFPMAQVHQGLMPTA
SG 56 443-460 SEQ ID NO:57 MAQVHQGLMPTAPPEDPAVD SG 57 449-458 SEQ
ID NO:58 MPTAPPEDPAVDLLKNYMQL SG 58 457-476 SEQ ID NO:59
PAVDLLKNYMQLGKQQREKQ SG 59 465-484 SEQ ID NO:60
YMQLGKQQREKQRESREKPYK SG 60 473-493 SEQ ID NO:61
EKQRESREKPYKEVTEDLLH SG 61 482-501 SEQ ID NO:62
KPYKEVTEDLLHLNSLFGGDQ SG 62 490-510 The amino acid sequence for gag
of SIVmac239 is shown in SEQ ID NO:63. SEQ ID NO: 63 1 MGVRNSVLSG
KKADELEKIR LRPNGKKKYM LKHVVWAANE LDRFGLAESL 51 LENKEGCQKI
LSVLAPLVPT GSENLKSLYN TVCVIWCIHA EEKVKHTEEA 101 KQIVQRHLVV
ETGTTETMPK TSRPTAPSSG RGGNYPVQQI GGNYVHLPLS 151 PRTLNAWVKL
IEEKKFGAEV VPGFQALSEG CTPYDINQML NCVGDHQAAM 201 QIIRDIINEE
AADWDLQHPQ PAPQQGQLRE PSGSDIAGTT SSVDEQIQWM 251 YRQQNPIPVG
NIYRRWIQLG LQKCVRMYNP TNILDVKQGP KEPFQSYVDR 301 FYKSLRAEQT
DAAVKNWMTQ TLLIQNANPD CKLVLKGLGV NPTLEEMLTA 351 CQGVGGPGQK
ARLMAEALKE ALAPVPIPFA AAQQRGPRKP IKCWNCGKEG 401 HSARQCRAPR
RQGCWKCGKM DHVMAKCPDR QAGFLGLGPW GKKPRNFPMA 451 QVHQGLMPTA
PPEDPAVDLL KNYMQLGKQQ REKQRESREK PYKEVTEDLL 501 HLNSLFGGDQ
The following peptides are located within SEQ ID NO:63: p17 (1-135
SG 1-16); p27 (136-354 SG 17-43); x peptide (355-371 SG 44-45); p9
(372-447 SG 46-65); and, p6 (448-510 SG 56-62).
TABLE-US-00008 TABLE 8 Overlapping peptides in Env of SIVmac239
(25-mer with 13-mer overlapping) SEQ ID NO:64
MGCLGNQLLIAILLLSVYGIYCTL SE1 1-25 Y SEQ ID NO:65
LLLSVYGIYCTLYVTVFYGVPAWR SE2 13-37 N SEQ ID NO:66
YVTVFYGVPAWRNATIPLFCATKN SE3 25-49 R SEQ ID NO:67
NATIPLFCATKNRDTWGTTQCLPD SE4 37-61 N SEQ ID NO:68
RDTWGTTQCLPDNGDYSEVALNVT SE5 49-73 E SEQ ID NO:69
NGDYSEVALNVTESFDAWNNTVTE SE6 61-85 Q SEQ ID NO:70
ESFDAWNNTVTEQAIEDVWQLFET SE7 73-97 S SEQ ID NO:71
QAIEDVWQLFETSIKPCVKLSPLC SE8 85-109 I SEQ ID NO:72
SIKPCVKLSPLCITMRCNKSETDR SE9 97-121 W SEQ ID NO:73
TMRCNKSETDRWGLTKSITTTAST SE10 109-133 SEQ ID NO:74
WGLTKSITTTASTTSTTASAKVDM SE11 121-145 V SEQ ID NO:75
TTSTTASAKVDMVNETSSCIAQDN SE12 133-157 C SEQ ID NO:76
VNETSSCIAQDNCTGLEQEQMISC SE13 145-169 K SEQ ID NO:77
CTGLEQEQMISCKFNMTGLKRDKK SE14 157-181 K SEQ ID NO:78
KFNMTGLKRDKKKEYNETWYSADL SE15 169-193 V SEQ ID NO:79
KEYNETWYSADLVCEQGNNTGNES SE16 181-205 R SEQ ID NO:80
VCEQGNNTGNESRCYMNHCNTSVI SE17 193-217 Q SEQ ID NO:81
RCYMNHCNTSVIQESCDKHYWDAI SE18 205-229 R SEQ ID NO:82
QESCDKHYWDAIRFRYCAPPGYAL SE19 217-241 L SEQ ID NO:83
RFRYCAPPGYALLRCNDTNYSGFM SE20 229-253 P SEQ ID NO:84
LRCNDTNYSGFMPKCSKVVVSSCT SE21 241-265 R SEQ ID NO:85
PKCSKVVVSSCTRMMETQTSTWFG SE22 253-277 F SEQ ID NO:86
RMMETQTSTWFGFNGTRAENRTYI SE23 265-289 Y SEQ ID NO:87
FNGTRAENRTYIYWHGRDNRTIIS SE24 277-301 L SEQ ID NO:88
YWHGRDNRTIISLNKYYNLTMKCR SE25 289-313 R SEQ ID NO:89
LNKYYNLTMKCRRPGNKTVLPVTI SE26 301-325 M SEQ ID NO:90
RPGNKTVLPVTIMSGLVFHSGPIN SE27 313-337 D SEQ ID NO:91
MSGLVFHSQPINDRPKQAWCWFGG SE28 325-349 K SEQ ID NO:92
DRPKQAWCWFGGKWKDAIKEVKQT SE29 337-361 I SEQ ID NO:93
KWKDAIKEVKQTIVKHPRYTGTNN SE30 349-373 T SEQ ID NO:94
IVKHPRYTGTNNTDKINLTAPGGG SE31 361-385 D SEQ ID NO:95
TDKINLTAPGGGDPEVTFMWTNCR SE32 373-397 G SEQ ID NO:96
DPEVTFMWTNCRGEFLYCKMNWFL SE33 385-409 N SEQ ID NO:97
GEFLYCKMNWFLNWVEDRNTANQK SE34 397-421 P SEQ ID NO:98
NWVEDRNTANQKPKEQHKRNYVPC SE35 409-433 H SEQ ID NO:99
PKEQHKRNYVPCHIRQIINTWHKV SE36 421-445 G SEQ ID NO:100
HIRQIINTWHKVGKNVYLPPREGD SE37 433-457 L SEQ ID NO:101
GKNVYLPPREGDLTCNSTVTSLIA SE38 445-469 N SEQ ID NO:102
LTCNSTVTSLIANIDWIDGNQTNI SE39 457-481 T SEQ ID NO:103
NIDWIDGNQTNITMSAEVAELYRL SE40 469-493 E SEQ ID NO:104
TMSAEVAELYRLELGDYKLVEITP SE41 481-505 I SEQ ID NO:105
ELGDYKLVEITPIGLAPTDVKRYT SE42 493-517 T SEQ ID NO:106
IGLAPTDVKRYTTGGTSRNKRGVF SE43 505-529 V SEQ ID NO:107
TGGTSRNKRGVFVLGFLGFLATAG SE44 517-541 S SEQ ID NO:108
VLGFLGFLATAGSAMGAASLTLTA SE45 529-553 Q SEQ ID NO:109
SAMGAASLTLTAQSRTLLAGIVQQ SE46 541-565 Q SEQ ID NO:110
QSRTLLAGIVQQQQQLLDVVKRQQ SE47 553-577 E SEQ ID NO:111
QQQLLDVVKRQQELLRLTVWGTKN SE48 565-589 L SEQ ID NO:112
ELLRLTVWGTKNLQTRVTAIEKYL SE49 577-601 K SEQ ID NO:113
LQTRVTAIEKYLKDQAQLNAWGCA SE50 589-613 F SEQ ID NO:114
KDQAQLNAWGCAFRQVCHTTVPWP SE51 601-625 N SEQ ID NO:115
FRQVCHTTVPWPNASLTPKWNNET SE52 613-637 W SEQ ID NO:116
NASLTPKWNNETWQEWERKVDFLE SE53 625-649 E SEQ ID NO:117
WQEWERKVDFLEENITALLEEAQI SE54 637-661 Q SEQ ID NO:118
ENITALLEEAQIQQEKNMYELQKL SE55 649-673 N SEQ ID NO:119
QQEKNMYELQKLNSWDVFGNWFDL SE56 661-685 A SEQ ID NO:120
NSWDVFGNWFDLASWIKYIQYGVY SE57 673-697 I SEQ ID NO:121
ASWIKYIQYGVYIVVGVILLRIVI SE58 685-709 Y SEQ ID NO:122
IVVGVILLRIVIYIVQMLAKLRQG SE59 697-721 Y SEQ ID NO:123
YIVQMLAKLRQGYRPVFSSPPSYF SE60 709-733 Q SEQ ID NO:124
YRPVFSSPPSYFQQTHIQQDPALP SE61 721-745 T SEQ ID NO:125
QQTHIQQDPALPTREGKERDGGEG SE62 733-757 G SEQ ID NO:126
TREGKERDGGEGGGNSSWPWQIEY SE63 745-769 I SEQ ID NO:127
GGNSSWPWQIEYIHFLIRQLIRLL SE64 757-781 T SEQ ID NO:128
IHFLIRQLIRLLTWLFSNCRTLLS SE65 769-793 R SEQ ID NO:129
TWLFSNCRTLLSRVYQILQPILQR SE66 781-805 L SEQ ID NO:130
RVYQILQPILQRLSATLQRIREVL SE67 793-817 R SEQ ID NO:131
LSATLQRIREVLRTELTYLQYGWS SE68 805-829 Y SEQ ID NO:132
RTELTYLQYGWSYFHEAVQAVWRS SE69 817-841 A SEQ ID NO:133
YFHEAVQAVWRSATETLAGAWGDL SE70 829-853 W SEQ ID NO:134
ATETLAGAWGDLWETLRRGGRWIL SE71 841-865 A SEQ ID NO:135
WETLRRGGRWILAIPRRIRQGLEL SE72 853-877 TLL
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.sup.+ anti-CD4.sup.+. The other
duplicate set was stained with anti-CD3.sup.+ anti-CD8.sup.+ (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.sup.+ CD4.sup.+ and CD3.sup.+ CD8.sup.+ 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.
[0175] The frequency of CD4.sup.+ 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 (17 pools) and --SIV gag peptides (16
pools) were determined. Also determined was the frequency of
CD8.sup.+ 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 and SIV gag peptides. 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.sup.+ T cell responses as measured by IFN-g synthesis
in previously primed BALB/c mice.
EXAMPLE 4
Direct Delipidation of HIV-1 and Removal of Solvents with Charcoal
Column and Retention of HIV Proteins
[0176] 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. A charcoal column (0.5-ml) 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. The virus-solvent mixtures were loaded
individually onto separate columns. The columns were chased with 1
ml of PBS. The elution volumes were measured and samples assayed
for p24 by ELISA, protein, and subjected to Western blotting.
[0177] The samples treated with 1% DIPE showed excellent p24
recovery compared to controls. The samples treated with 10% DIPE or
butanoUDIPE 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.
[0178] 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
[0179] Reagents for comparison included delipidated SIVmac251, heat
inactivated SIV mac251 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-HCl, pH 7.4; 1% NP40; 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).
[0180] 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.
[0181] 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.
[0182] 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
Development of a Modified SARS Viral Particle for use as a
Vaccine
A. Optimization of a Solvent Treatment Method for SARS Virus
[0183] Seed virus production of virus. Stock SARS virus (specimen
number 809940 strain 200300592) was obtained from the Centers for
Disease Control (CDC). The virus is grown in Vero E6 cells (ATCC
CRL 1586). The virus sample is thawed and 0.1 ml is inoculated with
a pipette into each of 5 test tubes of Vero E6 cells containing
about 2 ml outgrowth medium (90% Eagle's minimal essential medium
in Earle's balanced salt solution with 10% fetal bovine serum). The
remainder of the virus sample is stored at -80.degree. C. When
75-100% of the cell sheet in each tube show cytopathic effects
(CPE), the cells are harvested by freezing and scraping, pooled and
frozen at -80.degree. C. in 1 ml aliquots. The virus is titered in
test tubes of Vero E6 cells by the TCID.sub.50 method (serial 1:10
dilutions of virus in quadruplicate). Solvent treatment of virus.
SARS virus is solvent-treated by various methods used for SIV, DHBV
and BVDV as described herein, to optimize the process for maximum
envelope protein recovery and minimum residual infectivity.
Parameters explored for SARS virus solvent treatment are: solvent
type or combinations; solvent ratios; solvent to virus ratio;
treatment time; treatment temperature; mixing method; and solvent
removal process. Stock SARS virus preparations in PBS (phosphate
buffered saline) are combined with DIPE resulting in about 2000 to
10,000 ppm and mixed by end over end rotation for 20 to 60 minutes
followed by centrifugation at 1000.times.g for 2 minutes. Residual
solvent is removed by either vacuum evaporation or adsorption to
activated charcoal. In addition, combinations of DIPE and n-Butanol
are tested in ratios of 60:40 to 95:5 (vol/vol), resulting in about
200 to 40,000 ppm total solvent concentration, mixed end over end
for 20 to 60 minutes followed by centrifugation at 1000.times.g for
2 minutes. Residual solvent is removed by adsorption to activated
charcoal.
[0184] All samples from the various treatment methods described
above are characterized by PAGE, including Western blot, to
determine presence of viral protein and total protein.
Quantification of specific viral antigens and proteins are
evaluated by immunospecific assay such as ELISA. Infectivity is
evaluated using Vero E6 cytopathic assay (Reed and Muench; Am. J.
Hygiene 1938; 27:493-497). Selection is made as to the most
effective method of solvent treatment based on maximum target viral
protein recovered, greatest reduction in infectivity and
immunogenicity in mice.
B. Optimization of a Chemical Treatment Method for SARS Based on
Known Viral Inactivation Agents
[0185] In situations where the present treatment method reduces
infectivity to a level that is insufficient for a vaccine, chemical
inactivation of the solvent-treated virus may be indicated.
Chemical inactivation is considered successful if infectivity is
reduced by 6 logs.
Methods. The light-activated cross-linking reagent psoralen is
used. The psoralen tricyclic planar ring system intercalates into
single stranded RNA and is light activated. NHS-psoralen (Pierce
Biochemicals, Rockford Ill.) is dissolved in DMSO before adding to
aqueous reaction mixture. The NHS ester cross-links to primary
amines at pH 7-9. Solvent-treated virus solution is mixed with
NHS-psoralen (150 mM) in 0.1M sodium phosphate, 0.15M NaCl, pH 7.2.
Photoreactive coupling is achieved by exposure to light >350 nm
for 30 minutes or 3 Joules/cm.sup.2.
[0186] Cytopathic endpoints (CPE) in Vero E6 cells is typically
noted on the fifth day post-inoculation. It is focal in appearance,
with cell rounding and a refractiveness in the affected cells that
is followed by cell detachment. The CPE quickly spreads to involve
the entire cell monolayer within 24-48 hours. Thus if cell
integrity is destroyed it indicates that the virus is
infectious.
C. Evaluation of Native Viral Protein Structure and Viral Envelope
Changes Post Treatment
[0187] To evaluate the effect of the solvent treatment on viral
proteins, virus samples are characterized by non-denaturing PAGE
including Western Blot to determine presence of native viral
protein. Total soluble protein is measured using SDS PAGE. The most
effective method of solvent treatment is selected based on maximum
target viral protein recovered and greatest reduction in
infectivity. A double antibody sandwich ELISA is used to detect
SARS antibodies (Current Protocols in Immunology, Vol 1, supp. 8,
1991, John E Coligan, et al. eds.; Richard Coico, series ed.,
publisher: Current Protocols, John Wiley and Sons). Polyclonal
anti-SARS antibody is biotinylated and SARS virus antigen is
produced from stock SARS virus.
Native gel electrophoresis. Native gel electrophoresis is performed
at room temperature in polyacrylamide gels and proteins are
visualized either with silver staining or are transferred to
nitrocellulose for detection with labeled goat-anti-mouse
antibodies (Western blot). Samples of SARS virus pre and post
solvent treatment are analyzed using a pool of SARS virus proteins
as a standard. Western blot. Proteins on gels are transferred to
nitrocellulose membranes. For high molecular weight proteins
transfer time is at least 90 minutes. After blocking with BSA and
milk, nitrocellulose is incubated with polyclonal antibodies to
SARS virus spike and membrane proteins. Mouse antibodies are
visualized with horseradish peroxidase conjugated goat anti-mouse
antibodies. Commercially available SARS virus polyclonal antibodies
are purchased. Alternatively, the antibodies are produced in
weanling BALB/c mice by the method briefly described below.
Production of mouse anti-SARS antibodies. If SARS polyclonal
antibodies are not commercially available, mice are injected with
concentrated psoralen-treated stock virus preparation that has been
purified by sucrose density gradient centrifugation. Inactivation
is confirmed in Vero E6 cells. Twenty-two weanling BALB/c mice are
divided into 2 groups of 8 mice each with the remaining 6 mice as
controls. The two groups of 8 mice each are inoculated
subcutaneously (sc) with 10 ug (low) or 50 ug (high) doses of the
virus prep mixed with MPL (monophosphoryl lipid A, synthetic
trehalose dicorynomycolate; Ribi Adjuvant System, Corixa Corp.
Hamilton, Mont.). The 6 control mice are inoculated with an
equivalent amount of the cell culture medium mixed with adjuvant.
Inoculations are repeated at 2 and 4 weeks. At 6 weeks mice are
anesthetized and exsanguinated by retro-orbital
bleeding+intracardiac puncture. The serum from each group is pooled
to titer for neutralizing antibody.
[0188] If SARS virus spike and membrane proteins are in their
native conformation, antibodies raised to these intact proteins in
mice are recognized in the Western blot. The silver stained gels
are expected to show retention of viral proteins until the point
where solvent treatment denatures the proteins such that they can
no longer be detected by this method.
Additional and alternative methods. Additional methods are used to
confirm results from Western blots. Electron microscopy is used to
assess virus structural integrity and to compare changes pre and
post solvent treatment (Graham D R, et al., (2003) J. Virol.
77(15): 8237-8248). Viruses are inactivated with glutaraldehyde
prior to removal from the BSL-3 facility.
EXAMPLE 6
Ability of Solvent and Chemically Treated SARS Viral Particles to
Produce an Immune Response in Mice
[0189] Animals are vaccinated with viral preparations from solvent
treatment methods using varying concentrations of solvents, mixing
times and energy as well as solvent combinations resulting in low
to high degrees of lipid removal. Comparison of results from each
method in the vaccinated animals is used to determine which viral
prep provides the best immunological response. To be useful as a
vaccine the solvent-treated SARS virus must be both antigenic, as
evidenced by antibody production and cause increased cytokine
production.
A. Injection of Mice with Solvent and Chemically Treated Sars Viral
Particles for Antibody Production and to Test for the Elicitation
of Neutralizing Antibodies
[0190] Previously primed Balb/c mice are used to determine the
minimal dose of solvent-treated SARS virus that leads to readily
recognizable virus specific humoral or cellular immune response in
these mice using methods described by Ansari A., et al. (J.
Virology 76 (4): 1731-1743, 2002). Twenty adult female Balb/c mice
are each injected with 25 ug of chemically inactivated SARS virus
protein incorporated in an equal volume of adjuvant subcutaneously.
Four mice serve as control non-immunized mice (Group 1).
[0191] Sufficient SARS virus is treated according to methods
described in Example 5 so that the amount needed for boosting these
mice per schedule is available. On day 14 following initial
priming, 5 groups of 4 mice per group are treated as follows: Group
2--0.5 ml saline, Group 3--0.5 ml saline containing 10 ug of
solvent-treated virus, Group 4--0.5 ml saline containing 1 ug of
solvent-treated virus, Group 5--0.5 ml saline containing 0.1 ug of
solvent-treated virus, Group 6--0.5 ml saline containing 0.01 ug of
solvent-treated virus. Four days after boosting, all mice are
anesthetized and blood is collected via retro-orbital puncture.
Serum is obtained from the collected blood. Spleens are collected
from each test mouse for spleen cell preparation (see below). Serum
and spleen cells collected from these mice are used as the basis
for the analyses as described below in this example.
B. Test for Production of Mouse Neutralizing Antibodies in Serum
Using Vero E6 Cell Cytopathic Assay
[0192] To determine if the treated virus preparations are capable
of raising SARS virus neutralizing antibodies serum samples
collected from the mouse immunization are tested to evaluate if
they are capable of protecting Vero E6 cells from cytolysis.
Purification of virions. Briefly, viruses are isolated from
clarified cell culture supernatants by two successive rounds of
ultracentrifugation in 25 to 50% sucrose density gradients.
Virus-containing fractions are identified by UV absorption at 254
and 280 nm. Peak UV-absorbing fractions are pooled, diluted to
below 20% sucrose with TNE buffer (0.01 M Tris-HCl [pH 7.2], 0.1 M
NaCl, and 1 mM EDTA), ultracentrifuged to a pellet, and resuspended
in TNE buffer. Samples are stored at -80.degree. C. Treated virus
is prepared by incubating virus at the indicated concentration of
capsid protein in the presence of the appropriate agent under the
appropriate incubation conditions. Virus is then repurified through
a 20% sucrose pad by ultracentrifugation for 1 h at 100,000.times.g
at 4.degree. C. Virus Neutralization Assay. Stock SARS virus
obtained from the CDC is titrated in quadruplicate in test tubes of
freshly confluent Vero E6 cells for 7 days at 37.degree. C. to
obtain the TCID.sub.50/0.1 ml based on the appearance of CPE. The
inactivated mouse anti-SARS antiserum is serially diluted 1:10
using cell culture medium without serum. Equal volumes of diluted
specific antiserum are mixed with 100 TCID.sub.50 of stock SARS
virus and incubated for 1 hour. Duplicate tubes of Vero E6 cells
are inoculated with 0.2 ml of each virus-antiserum dilution mixture
and incubated for 7 days. This titration is repeated with each
neutralization assay. The dilution of antiserum that neutralizes at
least 100 TCID.sub.50 of virus, based on the appearance of CPE,
represents one antibody unit. In additional neutralization assays,
serial 1:10 dilutions of the virus to be confirmed as SARS and
twenty antibody units of specific immune serum are employed in
equal volumes. Infectivity assay. Each solvent-treated sample of
SARS virus is inoculated into two or four tubes of Vero E6 cells
and incubated for at least 7 days to detect the presence of CPE.
Non-solvent-treated stock SARS virus is inoculated as above as a
control. Virus titers are calculated by TCID.sub.50. It is expected
that the SARS virus causes cells to round up, become refractive and
detach in 2448 hours. If neutralizing antibody is present, the
cells remain intact. Neutralizing antibody in the test sera should
protect cells from 100 TCID.sub.50 of virus. If mouse antibodies to
Vero cell proteins are produced, serum from mice injected with mock
viral preparations starting with Vero E6 cells is used as a
control. If necessary, anti-Vero cell antibodies are removed from
mouse sera by affinity purification. C. Evaluate Mouse Cellular
Response on Vaccination with Solvent-Treated SARS Viral
Particles
[0193] Cytokines are critical in orchestrating immune responses. A
cellular response is significant relative to addressing the issue
of transient immunity seen with other coronavirus vaccines. As an
indication of mouse cellular immune response, the cytokine gamma
interferon, and interleukins such as IL-2, are measured as used for
retroviruses in vaccinated mice from the method described above in
this example.
Collection of Spleen Cells and Intracellular Cytokine Staining
Analysis. Spleen cells are collected aseptically and a single cell
suspension made, by forcing through a narrow gauge needle. Cell
counts are performed. Cells are resuspended at 1 million cells/ml
in RPMI 1640 complete media (RPMI 1640+100 U/ml penicillin+100
ug/ml streptomycin+2 mM L-glutamine +10% select lot of fetal calf
serum). Cell suspension (100,000 cells) is dispensed into wells of
a 96-well plate. Media is added to triplicate wells (negative
control) and phorbol myristic acetate (PMA 50 ng/ml)+Ionomycin (1
ug/ml) to 3 additional wells (positive control). The SARS pools of
overlapping peptides (set up as a grid) to cover certain SARS
coding sequences for viral structural genes (the E, M and S protein
sequences) is then added to the appropriate wells. The media
cocktail is added and incubated overnight. Add, incubate, remove
and wash as appropriate for additions of BrefeldinA solution,
antibody cocktail of PerCP-labeled CD4 and FITC-labeled CD8 in FACS
wash. The contents of each well are transferred to FACS tubes
followed by addition of perm/fix. After wash with Perm Wash, add
phycoerythrin (PE) anti human IFN-gamma. Repeat incubation, wash
and remove wash solution. Fresh 1% paraformaldehyde is added and
samples are refrigerated in the dark until ready to analyze. The
data on all samples is collected and the thresholds are drawn based
on the signal obtained with the media control and PMA+Ionomycin.
Data from on about 100,000 events is collected. The peptides are
identified that induce a positive interferon gamma or interleukin
response to overlapping peptides. The presence of cytokine positive
cells indicate that the solvent-treated SARS virus is effective in
eliciting a cellular immune response.
EXAMPLE 7
Delipidated SIV Virus Shows Reduced Infectivity and Causes
CD4.sup.+ and CD8.sup.+ T-Cell Immunological Responses When
Administered to Mice
[0194] A prime-boost immunization strategy using SIV delipidated
pursuant to the present invention gives rise to a broader CD4.sup.+
and CD8.sup.+ T-cell responses (interferon gamma production) in
mice than aldrithiol-2 (AT-2) treated or live virus. More
specifically, the present invention gives rise to an improved
immunological response across a broader array of antigens as
compared to non-delipidated viral particles. The present invention
specifically encompasses a modified viral particle having an
increased immunological response to a wider range of antigens, such
as a range of a minimum of 5% more antigens as compared to
non-delipidated viral particles.
[0195] In the present example, the delipidation of SIVmac251
reduced viral infectivity while retaining the major SIV proteins
(env, gag, pol, tat). The studies were carried out in Balb/c mice
immunized with AT-2-treated virus subcutaneously (sc) plus adjuvant
and boosted with either AT-2-treated virus, live virus or
delipidated virus. Routes of administration and intervals between
prime and boost and dose levels were evaluated. Spleen cells were
collected and cultured with individual pools of overlapping SIV env
and gag peptides covering the entire SIV amino acid sequence for
env and gag. The ability of the spleen cells to synthesize
(interferon) IFN-gamma by standard intracellular cytokine staining
(ICC) and flow cytometry was measured. Delipidation was performed
using 1% DIPE.
Materials and Methods: SIVmac251 Antigen Treatments
[0196] AT-2 inactivation: For the purpose of primary immunization
as well as boosting control, aliquots of sucrose banded SIVmac251
were inactivated via treatment with AT-2 as described previously
(Rossio et al., J. Virol. 72: 7992, 1998). Briefly, a 100 mM stock
solution of AT-2 (Aldrich, Milwaukee, Wis.) was prepared freshly in
dimethyl sulfoxide (DMSO). AT-2 was then added directly to the
virus at a final concentration of 300 .mu.M and incubated for 1 h
at 37.degree. C. before aliquoting the virus and storing it at
-70.degree. C., until used for immunization. DIPE solvent
treatment: Two hundred fig aliquots of sucrose purified SIVmac251
total protein were diluted in phosphate buffered saline (PBS) and
added various amounts of diisopropyl ether (DIPE) (VWR, West
Chester, Pa.) to bring the total volume to 1 mL in Eppendorf
microfuge tubes to achieve various DIPE concentrations. Solvent
treatment of the viral antigen preparation was performed for 20
minutes at room temperature. After a brief centrifugation to
collect the sample to the bottom of the tubes, the solvent was
evaporated in a Speedvac evaporator (Savant) for 90 minutes at room
temperature. At the end of this procedure, the volume was
reconstituted to 1 ml using injection grade water. A 25 .mu.L of
solvent-treated sample was diluted with 75 .mu.L distilled water
and submitted to gas chromatography analysis to ascertain removal
of solvent. The acceptable limit of residual DIPE solvent in any
sample used for immunization was .ltoreq.25 ppm. Each sample was
then aliquoted in appropriate quantity for booster immunization and
stored at -70.degree. C.
Viral Protein Recovery and Infectivity Assays
[0197] The effect of solvent treatment of SIVmac251 was ascertained
by total protein analysis using the BCA (Pierce, Rockford, Ill.)
and the Lowry assay (Biorad, Hercules, Calif.), polyacrylamide gel
electrophoresis followed by silver staining and by Western blot
analysis using a pool of SIV reactive monkey serum. In addition,
SIVgag p27 recovery was tested by EIA (Coulter Immunotech, Hialeh,
Fla.) and viral RNA by real-time amplification (Amara et al.,
Science 92:69, 2001). Residual viral infectivity was evaluated in
each treated aliquot by standard titration on CEMx174 cells and
monitoring of p27 production in the supernatant fluids of
individual well. The infectious titers were calculated according to
the Spearman-Karber method.
Isopycnic Density Gradient Centrifugation
[0198] Virus density profiles were evaluated by subjecting them to
isopycnic gradient centrifugation. Briefly, 1.3 ml each of 20%-60%
sucrose in phosphate buffered saline (PBS) was over-layered, with
8% increments in sucrose concentrations. Six sucrose concentrations
were layered, from 60% sucrose at the bottom, to 20% sucrose at the
top. Virus samples (prepared after pelleting through a 20% sucrose
cushion) in 750 .mu.l PBS were carefully over-layed on top of the
20% sucrose. All tubes were spun in an 80Ti rotor for the Beckman
L8 Ultracentrifuge at 40,000 rpm, and at 4.degree. C. for 16 h.
Starting from the top, 17 fractions of 525 .mu.l per tube were
collected. Virus concentrations were analyzed using a commercial
SIV Gag p27 ELISA kit (Coulter, Calif.).
Fast Performance Liquid Chromatography (FPLC) Virus Analysis
[0199] Delipidated viruses were further analyzed by FPLC in a
Pharmacia FPLC System. Virus samples (200 .mu.l) were injected into
a Superose 6 HR 10/30 (Pharmacia, Sweden) column. Sixty fractions
of 500 ul each were collected, at a flow rate of 0.4 ml/min in PBS
without Ca and Mg. Presence of SIV in the fractions was detected by
a p27 ELISA (Coulter, Calif.). Amounts of cholesterol in the virus
fractions were analyzed by the Amplex Red Total Cholesterol Assay
according to manufacturer's instructions (Molecular Probes,
OR).
Immunization of Mice
[0200] Four to six-10 week old female Balb/C mice were given a
primary immunization with 10 .mu.g of sucrose banded AT-2
inactivated SIVmac251 (ABI, Columbia, Md.) emulsified in Freund's
incomplete adjuvant (IFA) and administered subcutaneously (sc). For
purposes of control some mice were primed with IFA only. Groups of
6 animals were then administered a booster immunization 2 weeks
later using variable doses of treated vs. non treated SIVmac251
intravenously. The animals were then sacrificed 4 days post boost
to collect blood and splenocytes to perform the immune analyses
described below.
Intracellular IFN-.gamma. Response Evaluation of Cell Mediated
Responses
[0201] These analyses were performed using intracellular cytokine
(ICC) analyses following short-term antigen specific restimulation
in the presence of 5 .mu.g/mL of Brefeldin A and 1 .mu.g/ml each of
anti-mouse CD28 and CD49d monoclonal antibodies followed by
evaluation of the frequencies of IFN-.gamma. producing CD4.sup.+
and CD8.sup.+ T-cells. The standard protocol consisted of a 12 h
re-stimulation of 1.times.10.sup.6 splenocytes with pools of
peptides (containing 2 .mu.g/ml of each individual peptide)
encompassing the entire SIV gag (16 peptide pools, 20-mers
overlapping by 12 residues) and SIV env (17 peptide pools, 25-mers
overlapping by 13 residues), each pool containing 7-9 peptides.
Positive control samples consisted of splenocytes stimulated with
the mitogens PMA/ionomycin and PHA; negative controls are no
peptide stimulation and stimulation with the ovalbumin specific
peptide SYNFEKL (SEQ ID NO: 136). The cultures were carried out for
2 hours before adding the Brefeldin A designed to prevent excretion
of the cytokine and promote its intracellular accumulation. The
restimulated splenocytes were then stained for CD4.sup.+, CD8.sup.+
and intracellular for IFN-.gamma.. Evaluation of frequencies of
IFN-.gamma. positive CD4.sup.+ and CD8.sup.+ T-cells were analyzed
by counting about 200,000 events/sample using a FACS Calibur
(Beckton Dickinson, Mountain View, Calif.).
Serology
[0202] SIV EIA: Serum samples were titered for antibodies to viral
epitopes using routine EIA and Western Blot analysis. Briefly,
poly-L-Lysine (10 .mu.g per ml of PBS) coated ELISA micro plates
were adsorbed 2 .mu.g purified SIVmac251/well overnight in standard
bicarbonate coating buffer, pH 9.6 at 4.degree. C. Following 3
washes with PBS/Tween 20, the plates were blocked for 1 h at room
temperature with PBS containing 2% non-fat dry milk. Sequential
two-fold serum dilutions were then added to the plate as well as
positive and negative control samples in duplicates and incubated
at 37.degree. C. for 2 h. After washing the unbound antibodies, the
plates were incubated for 1 h at 37.degree. C. with an alkaline
phosphatase-anti mouse IgG conjugate (Southern Biotech, Birmingham,
Ala.), and later developed with p-nitrophenylphospbate (BioRad) at
room temperature. The plates were read at a 450 nm wavelength using
an ELISA reader (Molecular Devices, Sunnyvale, Calif.). SIV Western
blots: For Western blot analysis, commercially available SIV
western blot kits (Zeptometrix, Buffalo, N.Y.) were utilized
against mouse sera diluted 1:100 and developed according to the
manufacturer's instructions.
Results
[0203] Viral Delipidation Results in Removal of Cholesterol without
Loss of Viral Proteins
[0204] Our previous optimization procedures led to the finding that
DIPE treatment effectively delipidated HIV without significant loss
of viral proteins (data not shown). We extended these findings to
evaluate whether this method could delipidate SIV-mac251.
SIV-mac251 was delipidated using DIPE without significantly
affecting total protein or viral proteins (p27). Recoveries of both
total viral protein and viral gag p27 were not significantly
different when compared to live SIV. These findings were confirmed
by silver staining and Western blot analysis of SIV. Delipidated
virus showed a reproducible 2 log reduction in infectivity (FIG.
7). Removing cholesterol from virus using our method reduces
infectivity in a similar manner to .beta.-CD removal of cholesterol
in HIV-1 (Nguyan et al., J. Immunol. 168:4121, 2002; Graham et al.,
J. Virol. 77:8237, 2003), without losing viral RNA or viral
proteins. To further characterize the loss of lipids to the
physical properties of the treated virus, we evaluated the virus
particle profiles by fast performance liquid chromatography (FPLC)
(FIG. 5). The FPLC profiles of the control and aldrithiol-2 (AT-2)
treated viruses were similar (data not shown). However, DIPE
treated virions changed their structural profile, compared to the
live control virions. To evaluate whether our delipidation
procedure led to removal of cholesterol, we analyzed treated
viruses for cholesterol using the Amplex Red assay following FPLC
separation. The DIPE treated viruses had approximately 80% less
cholesterol than the control virus when expressed as
cholesterol/gag p27 protein ratio. Viruses were further analyzed by
isopycnic density gradient centrifugation, to evaluate particle
densities. Delipidation changed the buoyancy of the virions,
resulting in a shift of the density range of viral particles (FIG.
4).
Delipidated Viruses are Able to Elicit Broader Cell-Mediated Immune
Responses During Boosting
[0205] To evaluate whether the delipidated viruses had enhanced
immunogenicity in boosting cell mediated immune responses, we
boosted mice primed with AT-2 inactivated SIV (Rossio et al., J.
Virol. 72: 7992, 1998; Arthur et al., AIDS Res. Human Retroviruses
14:Suppl. 3. S311, 1998) with control and delipidated virus. After
two weeks, immunized mice groups (6 mice per group) were boosted
with 1 .mu.g total viral protein of either live SIV, AT-2
inactivated SIV, or DIPE delipidated SIV. T-cell responses were
evaluated using SIV Gag and SIV gp120 envelope overlapping peptide
pools, and responding cells detected by intracellular
interferon-.gamma. (IFN-.gamma.) flow cytometry (ICC). DIPE
delipidated virus booster elicited broader CD4.sup.+ and CD8.sup.+
responses, compared to control or AT-2 groups (FIGS. 8A and 8B).
Specific IFN-.gamma. peptides were also determined from the peptide
pool grids, yielding similar patterns to those seen when analyzing
the peptide pools. DIPE treated SIV also elicited new peptide pool
recognition patterns, compared to the other groups (Table 9). The
data were especially striking for CD4.sup.+ responses to env
peptide pools. DIPE group had a statistically significant increase
in responses compared to the live SIV boosted group (p=0.006), and
to the AT-2-treated SIV boosted group (p=0.0001). Similar trends
were observed with the DIPE treated SIV for CD8.sup.+ env peptide
pool responses (p=0.001 relative to live and p=0.02 relative to
AT-2 group). CD4.sup.+ gag responses were significantly increased
as well (p=0.03 relative to AT-2 group). The DIPE treated SIV
boosted group also had more IFN-.gamma. positive cells than the
other two groups. Antigen dosage studies indicated that a
surprisingly low dose of 1 .mu.g of DIPE delipidated virus (which
corresponds to approximately 200 ng of SIV p27) was sufficient to
elicit broad CD4.sup.+ and CD8.sup.+ immune responses to both gag
and env. Broad CD4.sup.+ and CD8.sup.+ responses to env and gag
peptide pools were observed in mice boosted with delipidated virus
when compared to AT-2 treated or live virus boost (p>0.001).
[0206] Predominantly CD4.sup.+ T cell responses were observed at
antigen doses as low as 0.05 ug of delipidated virus administered
IV without adjuvant, whereas higher doses were needed for AT-2 or
live SIV protein. Preliminary antibody responses indicate that the
delipidated virus is stimulating antibody responses as well. These
findings show a CD4.sup.+ and CD8.sup.+ cellular responses to a
broad array of SIV antigens elicited by very low boost
concentrations of virus delipidated with the method of the present
invention.
[0207] In the following few paragraphs a response is operationally
defined as a CD4 cellular response to SIV env peptides in terms of
a percentage of CD4.sup.+ cells that are positive for interferon
gamma. Peptide pools that elicited responses, and several ranges of
responses (percentage of CD4+ cells that are positive for
interferon gamma) are indicated.
[0208] The CD4 cellular response to SIV env peptides was not
significant in mice treated with 5 ug of live virus. Following
administration of various amounts of 1% DIPE delipidated virus, a
CD4 cellular response to SIV env peptides was observed. At a dose
of 0.05 ug, a response was elicited from three env peptide pools 5
(0.13-0.22%), 6 (-0.3-0.13%) and 13 (0.13-0.22%). At a dose of 1.0
ug, a broad response was elicited from over several env peptide
pools (3, 4, 5 (0.06-0.23%), 8, 11, 12 (0.19-0.45%), 13
(0.13-0.39%), 14 (0.13-0.34%), 15 (-0.03-0.24%)). At the higher
dose of 5 ug, a response was observed to env peptide pool 5
(0.17-0.23%).
[0209] The CD4 cellular response to SIV env peptide to boost with
various amounts of AT-2 treated virus revealed limited response. At
a dose of 0.05 ug, a response was elicited from one env peptide
pool (10 (0.17-0.25%)). At a dose of 1.0 ug, a response was
elicited from about one env peptide pool (10 (0.08-0.22%)). At the
higher dose of 5 ug, the CD4 cellular response was not
significant.
[0210] The CD4 cellular response to SIV env peptide to boost with
various amounts of live SIV virus showed a response at a dose of
0.05 ug from pools 1 (-0.05-0.23%), 8 (0.13-0.21%), 12 (0.11-0.21%)
and 14 (-0.03-0.25%). At a dose of 1.0 ug, a response was elicited
from three env peptide pools (8 (0.22-0.36%), 12 (0.12-0.58%) and
13 (-0.09-0.33%)). At the higher dose of 5 ug, the CD4 cellular
response was not significant.
[0211] In the following few paragraphs a response is operationally
defined as a CD8+ cellular response to SIV env peptides in terms of
a percentage of CD8+ cells that are positive for interferon gamma.
Peptide pools that elicited responses and several ranges of
responses (percentage of CD8+ cells that are positive for
interferon gamma) are indicated.
[0212] Following administration of various amounts of 1% DIPE
delipidated virus, a CD8 cellular response to SIV env peptides was
observed. At a dose of 0.05 ug, a response was elicited from two
env peptide pools 5 (0.22-1.22%) and 13 (0.43-0.92%). At a dose of
1.0 ug, a broad response was elicited from several env peptide
pools (2 (0.18-0.34%), 3 (-0.06-0.35%), 4 (-0.03-0.15%), 5
(0.06-0.25%), 9 (0.24-0.41%), 10 (0.34-0.87%), 11 (0.22-0.71%), 12
(0.19-0.53%), 13 (0.11-0.35%), 14 (0.19-0.32%), 15 (0.98-1.35%) and
16 (0.11-0.31%) At the higher dose of 5 ug, a response was observed
to env peptide pool 13 (0.27-0.41%), 14 (0.28-0.48%) and 15
(0.31-0.35%).
[0213] Following administration of various amounts of AT-2 treated
virus, a limited CD8 cellular response to SIV env peptides was
observed. At a dose of 0.05 ug, a CD8 cellular response, was
elicited from env peptide pool 16 (0.08-0.45%). At a dose of 1.0
ug, a response was elicited from env peptide pools 7 (0.18-0.33%)
and 16 (0.29-0.88%). At the higher dose of 5 ug, the CD8 cellular
response was not significant.
[0214] Following administration of various amounts of live SIV, a
limited CD8 cellular response to SIV env peptides was observed. At
a dose of 0.05 ug, a CD8 cellular response, was elicited from
peptide pools 1 (-0.05-0.23%), 8 (0.13-0.2%), 12 (0.11-0.21%) and
14 (-0.03-0.25%). At a dose of 1.0 ug, a response was elicited from
peptide pools 8 (0.22-0.36%), 12 (0.12-0.58%), and 13
(-0.02-0.33%). At the higher dose of 5 ug, the CD8 cellular
response was not significant.
[0215] In the following few paragraphs a response is operationally
defined as a CD4 cellular response to SIV gag peptides in terms of
a percentage of CD4+ cells that are positive for interferon gamma.
Peptide pools that elicited responses, and several ranges of
responses (percentage of CD4+ cells that are positive for
interferon gamma) are indicated.
[0216] Following administration of various amounts of 1% DIPE
delipidated virus, a CD4 cellular response to SIV gag peptides was
observed. At a dose of 0.05 ug, a response was elicited from gag
peptide pools 5 (0.22-1.22%) and 13 (0.43-0.92%). At a dose of 1.0
ug, a broad response was elicited from about five gag peptide pools
(3 (0.19-0.72%), 5 (0.15-0.71%), 7 (0.12-0.77%), 10 (0.19-0.92%),
and 15 (0.42-1.35%)). At the higher dose of 5 ug, the response
decreased to about four gag peptide pools 3 (0.12-0.49%), 5
(-0.04-0.48%), 10 (0.11-0.52%), 14 (-0.03-0.52%), and 15
(0.18-0.56%).
[0217] Following administration of various amounts of AT-2 treated
virus, a limited CD4 cellular response to SIV gag peptides was
observed. At a dose of 0.05 ug, a CD4 cellular response, was
elicited from three gag peptide pools (10 (0.19-0.59%), 11
(0.11-0.39%), and 13 (-0.03-0.31%)). At a dose of 1.0 ug, a limited
response was elicited from gag peptide pool 7 (-0.05-0.27%). At the
higher dose of 5 ug, the CD4 cellular response was not
significant.
[0218] Following administration of various amounts of live SIV
virus, a CD4 cellular response to SIV gag peptides was observed. At
a dose of 0.05 ug, a CD4 cellular response, was elicited from about
2 gag peptide pools (2 (0.59-1.23%) and 9 (0.34-1.1%)). At a dose
of 1.0 ug, a response was elicited from about four gag peptide
pools (2 (0.39-1.12%), 3 (0.11-0.51%), 6 (0.21-0.72%), and 9
(0.15-0.51%)). At the higher dose of 5 ug, a response was elicited
from about two gag peptide pools (2 (0.16-0.51%) and 6
(-0.05-0.23%)).
[0219] In the following few paragraphs a response is operationally
defined as a CD8 cellular response to SIV gag peptides in terms of
a percentage of CD8+ cells that are positive for interferon gamma.
Peptide pools that elicited responses, and several ranges of
responses (percentage of CD8+ cells that are positive for
interferon gamma) are indicated.
[0220] Following administration of various amounts of 1% DIPE
delipidated virus, a CD8 cellular response to SIV gag peptides was
observed. At a dose of 0.05 ug, a response was elicited from about
five gag peptide pools (2 (0.19-0.92%), 3 (0.19-0.94%), 4
(0.18-0.95%), 6 (0.28-0.49%), and 13 (0.29-0.88%)). At a dose of
1.0 ug, a response was elicited from about six gag peptide pools (2
(0.01-1.01%), 3 (0.03-0.49%), 6 (0.01-0.99%), 7 (0.02-0.37%), 10
(0.01-0.92%), and 15 (0.05-0.65%)) At the higher dose of 5 ug, a
response was elicited from about seven gag peptide pools (2
(0.11-0.37%), 3 (0.16-0.54%), 4 (0.18-0.91%), 5 (0.18-0.71%), 10
(0.13-0.23%), 14 (0.13-0.81%), and 15 (0.2-0.56%)).
[0221] Following administration of various amounts of AT-2 treated
virus, a CD8 cellular response to SIV gag peptides was observed. At
a dose of 0.05 ug, a CD8 cellular response, was elicited from five
gag peptide pools (10 (0.28-0.71%), 11 (0.3-0.91%), 12
(0.23-0.76%), 13 (0.15-0.61%), and 14 (0.19-0.72%)). At a dose of
1.0 ug, a response was elicited from about three gag peptide pools
(10 (0.01-0.73%), 11 (-0.02-1.1%), and 12 (-0.05-0.72%)). At the
higher dose of 5 ug, a response was elicited from about one gag
peptide pool (10 (0.07-0.27%).
[0222] Following administration of various amounts of live SIV
virus, a CD8 cellular response to SIV gag peptides was observed. At
a dose of 0.05 ug, a CD8 cellular response, was elicited from about
3 gag peptide pools (2 (0.28-0.92%), 9 (0.32-0.82%), and 15
(0.21-0.43%)). At a dose of 1.0 ug, a response was elicited from
about five gag peptide pools (2 (0.01-0.91%), 3 (0.03-0.67%), 6
(0.01-0.71%), 9 (-0.25-0.8%) and 12-0.05-0.39%)). At the higher
dose of 5 ug, a response was elicited from about three gag peptide
pools (2 (0.19-0.71%), 9 (0.19-0.53%), and 12 (0.04-0.87%)).
[0223] Taken together, these data demonstrate that mice immunized
with AT-2 treated SIV virus show enhanced immunological responses
to boosting with delipidated SIV virus when compared to boosting
with AT-2 treated virus or live SIV virus. The delipidated SIV
virus was more immunogenic than the AT-2 treated virus in terms of
the percentage of CD4.sup.+ and CD8.sup.+ with enhanced IFN-.gamma.
staining.
[0224] Our data indicate that delipidated viruses elicited strong
T-cell mediated immune responses, without the use of an adjuvant.
Increase in the breadth and strength of the overall cell-mediated
immune response was observed in the DIPE boosted mice group,
compared to the live and AT-2 treated groups. Tables 9 and 10
present a summary of these results.
TABLE-US-00009 TABLE 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
CD4 ENV POOLS RESPONDING 0.05 .mu.g DIPE + + 1 .mu.g DIPE 5 .mu.g
DIPE + CD8 ENV POOLS RESPONDING 0.05 .mu.g DIPE + + 1 .mu.g DIPE 5
.mu.g DIPE + + + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CD4 GAG
POOLS RESPONDING 0.05 .mu.g DIPE + + 1 .mu.g DIPE 5 .mu.g DIPE + +
+ + CD8 GAG POOLS RESPONDING 0.05 .mu.g DIPE + + + + + 1 .mu.g DIPE
5 .mu.g DIPE + + + + + + + + + TABLE 9. SIV gag and env peptide
pool responses for CD4.sup.+ and CD8.sup.+ T-cells in mice boosted
with 0.05, 1, or 5 .mu.g total protein. 1 million mouse PBMCs were
stimulated with different peptide pools as indicated, for 2 h.
After blocking protein secretion by Brefeldin A, anti-CD4 and
anti-CD8 antibodies were added, cells permeabilized and further
stained with anti-IFN-.gamma. Ab. Cells were subsequently analyzed
by FACS. Any responses above 0.1% of total cells positive for
IFN-.gamma. staining were considered as a positive response. Shaded
symbols represent DIPE treated viruses at 1 .mu.g dose.
TABLE-US-00010 TABLE 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
CD4 ENV POOLS RESPONDING 1% DIPE + LIVE + AT-2 CONTROLS No
detectable responses CD8 ENV POOLS RESPONDING 1% DIPE + + + LIVE +
+ AT-2 + CONTROLS No detectable responses 1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 CD4 GAG POOLS RESPONDING 1% DIPE + LIVE + + AT-2
CONTROLS No detectable responses CD8 GAG POOLS RESPONDING 1% DIPE +
+ + + LIVE + + + + AT-2 + + CONTROLS No detectable responses TABLE
10. Mice were immunized with 10 .mu.g of SIV incorporated in
Freund's incomplete adjuvant sc and 2 weeks later boosted iv with
varying concentration of DIPE treated SIV, AT-2 treated SIV or
untreated live SIV. Controls consisted of groups of mice primed
with saline but boosted with DIPE, AT-2 or untreated virus or
groups of mice primed with SIV but boosted with saline. Spleen
cells were assayed for response to pools of SIV env or SIV gag
overlapping peptides utilizing the ICC assay for CD4.sup.+ or
CD8.sup.+ T-cells synthesizing IFN-g, and denotes a net response
(response to media and irrelevant peptide was deducted) to the
appropriate peptide pool.
Antibody Titers are Enhanced in DIPE Treated SIV Boosted Group
[0225] Antibody (Ab) titers to whole virions were determined for
each group. Antibody titers to SIV gp120 were significantly lower
in the AT-2 boosted group compared to the DIPE boosted groups
(p=0.02) (FIG. 9). In general, DIPE boosted mice gave higher Ab
readings, compared to either the live, or AT-2 boosted groups, for
both SIV gp120 and SIV Gag (FIG. 10). When Ab titers were measured
in a subsequent experiment at 4 weeks, boosting was observed for
all groups (data not shown). Gag (p55) antibody titers, measured by
ELISA (absorbance at 450 nm), were higher in serum from mice
boosted with delipidated SIVmac251 than either live or AT-2 treated
SIV boosted groups. Western Blot analysis supported the antibody
ELISA data, as a broader p27 band was observed by the delipidated
SIV boosted serum, compared to live or AT-2 treated mouse serum.
This indicates a broader p27 epitope recognition by gag antibodies
from the delipidated SIV boosted mice. Maturation of antibody
response to both gag and env was observed when mice were boosted 4
weeks after priming compared to a 2 week boosting post prime. Route
of administration, subcutaneous (sc) or intravenous (IV), did not
affect antibody (ELISA) titers. A stronger correlation is seen
between CD4.sup.+ T-cell and antibody responses to both SIVmac251
gag and env proteins in mice boosted with delipidated virus
compared to live or AT-2 treated virus boosts.
Strong Correlation Between CD4 Responses and Antibody Responses
[0226] We further determined the impact of immunization by
comparing the CD4.sup.+ responses to gag and env peptide pools to
the antibody responses to recombinant gag and env. A strong
correlation was observed between the cellular responses (CD4) and
the humoral responses (antibody responses) (FIG. 11), indicating
additional benefits of enhanced cell mediated immune responses.
[0227] DIPE-treatment created a powerful cell mediated immune
response, and a good humoral response in the absence of an
adjuvant. Significantly, an effective boosting was achieved with as
little as 1 mg total viral protein of DIPE treated SIV,
representing about 200 ng of SIV p27.
[0228] Our ability to elicit virus peptide specific immune
responses with as little as 1 .mu.g of total virus protein was both
surprising and unexpected. This level of immune response achieved
with a single IV boost without co-administration of adjuvants
suggests that the biochemical nature of delipidated virus is
sufficiently altered to direct an efficient processing and
presentation, or recognition of a larger number of viral peptides
different from those elicited by live or AT-2-treated SIV.
[0229] In conclusion, we have compared the immunogenicity of live
SIV, AT-2 treated SIV, and delipidated SIV (DIPE) in Balb/c mice,
and observed a significant enhancement of cell-mediated immune
responses from the groups boosted with DIPE treated viruses.
Surprisingly, effective boosting was achieved with a very low dose
of 1 .mu.g total viral protein, which corresponds to about 200 ng
of SIV p27. These results were obtained without the use of
adjuvants in the boost doses, indicating a substantial increase in
immunogenicity. Our results show that delipidating viruses enhanced
the antigenicity of the virus, while significantly reducing its
infectivity. Our results differ from previous findings that
cholesterol-depletion of HIV dramatically reduces virus infectivity
(Nguyan et al., J. Immunol. 168:4121, 2002; Graham et al., J.
Virol. 77:8237, 2003; Liao et al., AIDS Res. Human Retroviruses
19:675, 2003), because .beta.-CD treated viruses resulted in
dramatic loss of viral RNA and viral proteins, thus contributing to
the loss of infectivity. Delipidated viruses have negligible loss
of viral RNA and viral proteins.
[0230] While not wanting to be bound by the following statement, it
is believed that the delipidation process may create virus
particles which are better processed or presented by antigen
presenting cells, leading to the broad peptide pool responses
observed. Additionally, delipidation of viruses could expose more
cellular antigens (picked up by the virus when budding from
infected CEMx174 cells) such as MHC II molecules, which could act
as adjuvants in enhancing cellular responses. Serum Ab titers and
Western blot analysis of Ab sera profiles, indicated enhanced
anti-Env antibodies, and consistent broadening of SIV gag specific
antibody responses in DIPE-treated SIV-boosted groups, perhaps
indicating an increase in anti-p27 Ab titers, or an increase in Ab
avidity to viral proteins. The present results demonstrate that
DIPE delipidation of SIV affects the immunogenicity of the virus in
mice. It is believed that this novel delipidation method will
contribute to HIV therapeutic vaccine design and development.
EXAMPLE 8
Total Protein and p24 Protein Recovery in HIV Virus Treated with
Various Delipidation Procedures
[0231] The applicants have found that the aforementioned
delipidation processes are capable of producing intact viral
particles, as measured by the degree of total protein recovery and
p24 protein recovery.
[0232] The sample containing the HIV virus was mixed with solvent
using end-over-end rotation at room temperature for 20 minutes at a
speed of 70%. Next the sample was centrifuged for 2 minutes at
1000.times.g and then passed through a charcoal column. Total
protein was measured by BioRad Assay. Viral p24 was measured by p24
sandwich ELISA (Coulter)
[0233] Total protein recovery for delipidation processes using 1%
DIPE, 1% butanol/DIPE, 1% butanol, 2% butanol, and 5% butanol are
within 10% of the control, specifically in the range of 63% to 75%
of total input. P24 protein recovery for delipidation processes
using 1% DIPE, 1% butanol/DIPE, 1% butanol, 2% butanol, and 5%
butanol are within 40% of the control, with 2% butanol yielding a
p24 protein recovery percentage of around 78% relative to a control
recovery percentage of around 83%.
EXAMPLE 9
Buoyant Density and Immunoreactivity (gp120 and p24) Profile of HIV
and SIV Particles Treated with Various Delipidation Procedures
[0234] The aforementioned delipidation processes modified the
buoyant density of viral particles. Changes in density are useful
indicators of successful delipidation because the removal of lipids
from viral particles changes the protein to lipid ratio and, as a
result, the particle density. In this experiment, the isopyknic
densities of control and solvent treated HIV and SIV particles were
determined and the changes in density were correlated with measured
lipid content of control and treated viruses.
[0235] Solvent treatments broadened the density range of HIV and
SIV particles and high solvent concentrations shifted the virus to
higher overall density, based on Western blot analyses and protein
profiles, which is consistent with the loss of lipids.
Specifically, FIG. 1 depicts the density of sucrose gradient
fractions as indicated by the graphing of density against fraction
number for viral particles subjected to delipidation using 1% DIPE,
1% butanol/DIPE, 1% butanol, 2% butanol, and 5% butanol, along with
a control group. HIV was delipidated and sucrose purified. Virus
was loaded onto sucrose gradients and centrifuged until equilibrium
densities were reached. FIG. 2 depicts the p24 protein
concentration for each of the fraction numbers. As expected, the
protein concentration for the control group was highest with 1%
butanol/DIPE demonstrating a relatively larger concentration of
p24, although registering at a higher density than the control.
Other density modified p24 concentrations were exhibited for 5%
butanol, 2% butanol, 1% butanol, and 1% DIPE. The density
modifications demonstrate a degree of success in delipidating the
viral particles.
[0236] The HIV-1 virus was run on a sucrose gradient and various
fractions were collected and then run on an SDS-PAGE gel,
transferred to a membrane, and blotted using a positive control
sera from an HIV-1 infected individual.
[0237] Western blot analyses were conducted with antibodies for
envelope protein gp120 and capsid protein p24 for the various
density fractions derived for each of the delipidation processes
and the control for HIV-1 viral particles. The Western blot
analysis of control samples revealed strong bands of p24 protein
and gp120 protein at the expected density fractions. The majority
of intact virions eluted in fractions 5-7. The various delipidation
processes produced changes in the location of the p24 and gp120
immunoreactive fractions, indicating alterations in the density of
the treated viral particles. Treatment of HIV-1 with 1% DIPE
produced a shift of immunoreactive bands to higher density
fractions. Treatment of HIV-1 with 1% DIPE/butanol and separately
with 1% butanol also produced a shift of immunoreactive bands to
higher density fractions. Treatment of HIV-1 with 2% butanol
resulted in a loss of many proteins, including a decrease in p24
protein and gp120 protein, and an increase in density of the viral
particles. Treatment of HIV-1 with 5% butanol resulted in an almost
complete loss of p24 protein and gp120 protein immunoreactivity,
and a marked increase in density of the viral particles.
[0238] In FIG. 3, an isopycnic gradient analysis of delipidated
HIV, indicated by a graphing of percent of total recovered p24
protein against fraction number, is shown. A substantial amount of
the total recovered p24 protein for the samples subjected to
delipidation processes is found at higher densities. For each of
the samples delipidated with 1% DIPE, 1% butanol/DIPE, 1% butanol,
2% butanol, and 5% butanol, greater amounts of p24 protein were
recovered at the higher fraction numbers (higher densities) as
compared to the control group. That density shift is further shown
in FIG. 4 where the isopycnic density of SIV-mac 251, indicated by
a graphic of gag p27 concentration against fraction number, is
depicted. Relative to a control, the delipidation samples for 1%
DIPE and 1% butanol both exhibited a shift in density.
EXAMPLE 10
Reduction in Cholesterol Content of HIV and SIV Viral Particles
Subject to Delipidation Procedures
[0239] The applicants have found that the aforementioned
delipidation processes modify the degree of cholesterol in viral
particles. Changes in cholesterol are useful indicators of
successful delipidation because the removal of lipids from viral
particles changes the amount of cholesterol and the cholesterol to
protein ratio. Exposure of HIV and SIV particles to organic
solvents removes lipids while preserving proteins, thereby
resulting in loss of viral infectivity while maintaining or
enhancing the immunogenicity of particles.
[0240] In Table 11 the cholesterol to total protein ratio of viral
particles delipidated by 1% DIPE, 1% butanol, 1% butanol/DIPE, 2%
butanol, and 5% butanol, along with a control, is shown. HIV was
delipidated and purified on 20% sucrose. Cholesterol was measured
with Amplex Red assay, a commercially available bioassay from
vendors such as Molecular Probes, Inc., and total protein was
measured. The data shows a decreased cholesterol content, relative
to total protein, for each of the delipidated samples.
TABLE-US-00011 TABLE 11 Cholesterol and protein levels in HIV
subject to different lipid removing solvents Chol. Protein Chol./
(.mu.g/ml) SD (ug/ml) % of Control (ug/ml) protein Control 11.06
0.31 100.00 75.45 0.15 1% DIPE 6.49 0.06 49.15 90.03 0.07 1%
But/DIPE 5.87 0.44 48.14 83.18 0.07 1% Butanol 5.52 0.60 45.90
82.08 0.06 2% Butanol 5.14 0.16 43.54 80.53 0.06 5% Butanol 3.86
0.07 35.08 75.01 0.05
[0241] SIV was delipidated and purified on 20% sucrose. Cholesterol
was measured with Amplex Red assay and Gag p27 protein measured.
Data is expressed as cholesterol to Gag p27 protein ratio. DIPE
treated virus had 80% less cholesterol than control, indicating
effective delipidation. Similarly, relative to the control, the 1%
DIPE sample has a decreased cholesterol to protein ratio. 1% DIPE
treatment effectively removed 80% cholesterol while maintaining the
structural integrity of the virus measured by the p27 recovery. 5%
DIPE:n-butanol treatment led to a dramatic loss of viral protein,
total protein, and cholesterol. This method was too harsh. 1%
butanol treatment was not effective at delipidating the virus, as
the amount of cholesterol measured was still intact. The recovery
of total cholesterol is about 37% and 78% for 1% butanol and 1%
DIPE, respectively, and the corresponding recovery of p27 protein
is about 90% and 15%, respectively, further indicating a successful
delipidation of viral particles while still keeping a substantial
portion of such viral particles intact. Referring to FIGS. 5 and 6,
FPLC profiles of fractionated SIV-mac251 are shown for Gag p27 and
cholesterol. The graphs demonstrate that, for a 1% DIPE
delipidation, the concentration of gag p27 substantially diverges
from the control at higher fraction numbers while the concentration
of cholesterol is substantially lower than the control for nearly
all fractions.
EXAMPLE 111
Monkeys Boosted with Delipidated HIV have Higher Ab Titers Compared
to Live HIV Boosted Group
[0242] Four monkeys were primed with an equivalent of 5 ug p24
HIV-IIIB in incomplete Freund's Adjuvant. Monkeys were then
separated into two groups of two monkeys. Group 1 (RIl & RFo)
received 1 ug DIPE delipidated HIV-IIIB every month; group 2 (RFt
& Rom) received 1 ug live HIV-IIIB every month. Cellular
parameters were measured by immunocytochemistry. Staining was done
at 7 days post boost, while Ab titers and neutralization Ab were
taken at 4 weeks post boosting. Ab titers to whole HIV-IIIB lysate
were measured. Group 1 animals (which received delipidated virus)
had higher Ab titers than the two control monkeys in Group 2.
Delipidated virus boosting enhanced Ab titers to the whole virion
(data not shown).
[0243] Pooled CD4 T-cell responses to all the peptide pools are
displayed in FIG. 12. Overall, animals showed a better response to
ENV peptide pools than to GAG peptide pools. Both of the animals in
Group 1 (RIl and RFo) had cumulative responses for Gag (>1.5%)
and for Env (>1.5%). Only one animal in the control Group 2
(RFt) had an appreciable response to Gag (>0.5%) and for Env
(>1.5%). The other control animal, Rom, had very low responses
to the peptide pools.
[0244] Overall, monkeys given delipidated virus showed better cell
mediated immune response (measured by ICC). The Ab data correlates
well with the CD4+ ICC data. Animals showing ICC responses also
have good Ab titers. The Western Blot data also correlates well
with both the Ab data and the ICC results.
EXAMPLE 12
Dendritic Cells Exposed to Delipidated SIV Stimulate Enhanced
Cd4.sup.+ Proliferation Compared to Dendritic Cells Exposed to Live
Virus
[0245] PBMCs from a long term non-progressor monkey were employed.
PBMCs were isolated using ficoll separation, and monocytes were
cultured out using plastic adherence of 3.times.10.sup.7 PBMC in 5
ml RPMI-10% FCS at 37.degree. C. for 2 hrs. Non-adherent cells were
removed and flasks gently washed with warm 1.times.PBS. Monocytes
were incubated with 1000 U/ml IL4 and 1000 U/ml of GM-CSF for 4
days in RPMI-15% FCS. This procedure generated immature dendritic
cells (DC).
[0246] Immature DC (2.times.10.sup.3) were pulsed with 50 ng of
AT-2 treated SIV, delipidated SIV (1% DIPE with end-over-end mixing
for 20 min) or live SIV for 3 hr at 37.degree. C. Cells were washed
extensively to eliminate excess virus and were checked by SIVp27
for amount of residual virus. DC (2.times.10.sup.3) were
resuspended for 3 days in R-15 with 100 U/ml TNF-a, IL4, GM-CSF to
induce DC maturation. Next, 2.times.10.sup.6 peripheral blood
lymphocytes (PBL) were added to the DC cultures, for 24-36 hr,
before performing proliferation assay using the cyQUANT Cell
Proliferation Assay Kit (Molecular Probes) [Note: CD8.sup.+ cells
were depleted from the PBLs prior to use]. Proliferation assay
performed according to manufacturer's protocol (cyQUANT-Molecular
Probes). Briefly, cells were pelleted and the supernatant removed.
The pellet was then frozen for about 1 hr, and 4.times. CyQUANT dye
concentration added to the pellet. The supernatant of lysed cells
was allowed to sit for about 10 min before reading a fluorescent
plate at wavelengths of 480 for excitation and 520 for
emission.
[0247] The % proliferation was calculated as follows: [(test
proliferation-control proliferation)/(control
proliferation)].times.100. The control proliferation is the
proliferation of PBMC+DC without adding the antigen to provide
background noise.
[0248] Dendritic cells (DC) are powerful antigen presenting cells
to the CD4, CD8, and CD20B-cells. The results demonstrate that
dendritic cells (DC) pulsed with delipidated SIV triggered a 16%
better proliferative response in CD4.sup.+ cells compared to DCs
pulsed with live virus (208672 with delipidated virus vs 165616
with live virus). This strongly suggests a better antigen
processing/presentation of the delipidated virus by the DC.
[0249] CD4 proliferation is a functional index of CD4 responses to
a given epitope. It is more specific readout than IFN-.gamma.
secretion, since in HIV infected people, their CD4 cells produce
IFN-.gamma., but do not proliferate in response to antigen.
[0250] Virus delipidated with the method of the present invention
can increase proliferation of antigen specific CD4.sup.+ cells
which leads to a more efficient maturation of the CD8.sup.+ cells
and maturation of plasma cells (B-cells which produce antigen
specific Ab). Since control of viral infection is dependent on
CD4.sup.+ cellular proliferation, the method of the present
invention provides an effective functional vaccine.
[0251] 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
136120PRTSimian Immunodeficiency Virus 1Met Gly Val Arg Asn Ser Val
Leu Ser Gly Lys Lys Ala Asp Glu Leu1 5 10 15Glu Lys Ile Arg
20220PRTSimian Immunodeficiency Virus 2Ser Gly Lys Lys Ala Asp Glu
Leu Glu Lys Ile Arg Leu Arg Pro Asn1 5 10 15Gly Lys Lys Lys
20320PRTSimian Immunodeficiency Virus 3Glu Lys Ile Arg Leu Arg Pro
Asn Gly Lys Lys Lys Tyr Met Leu Lys1 5 10 15His Val Val Trp
20420PRTSimian Immunodeficiency Virus 4Gly Lys Lys Lys Tyr Met Leu
Lys His Val Val Trp Ala Ala Asn Glu1 5 10 15Leu Asp Arg Phe
20520PRTSimian Immunodeficiency Virus 5His Val Val Trp Ala Ala Asn
Glu Leu Asp Arg Phe Gly Leu Ala Glu1 5 10 15Ser Leu Leu Glu
20620PRTSimian Immunodeficiency Virus 6Leu Asp Arg Phe Gly Leu Ala
Glu Ser Leu Leu Glu Asn Lys Glu Gly1 5 10 15Cys Gln Lys Ile
20720PRTSimian Immunodeficiency Virus 7Ser Leu Leu Glu Asn Lys Glu
Gly Cys Gln Lys Ile Leu Ser Val Leu1 5 10 15Ala Pro Leu Val
20820PRTSimian Immunodeficiency Virus 8Cys Gln Lys Ile Leu Ser Val
Leu Ala Pro Leu Val Pro Thr Gly Ser1 5 10 15Glu Asn Leu Lys
20920PRTSimian Immunodeficiency Virus 9Ala Pro Leu Val Pro Thr Gly
Ser Glu Asn Leu Lys Ser Leu Tyr Asn1 5 10 15Thr Val Cys Val
201020PRTSimian Immunodeficiency Virus 10Glu Asn Leu Lys Ser Leu
Tyr Asn Thr Val Cys Val Ile Trp Cys Ile1 5 10 15His Ala Glu Glu
201120PRTSimian Immunodeficiency Virus 11Thr Val Cys Val Ile Trp
Cys Ile His Ala Glu Glu Lys Val Lys His1 5 10 15Thr Glu Glu Ala
201220PRTSimian Immunodeficiency Virus 12His Ala Glu Glu Lys Val
Lys His Thr Glu Glu Ala Lys Gln Ile Val1 5 10 15Gln Arg His Leu
201319PRTSimian Immunodeficiency Virus 13Thr Glu Glu Ala Lys Gln
Ile Val Gln Arg His Leu Val Val Glu Thr1 5 10 15Gly Thr
Thr1420PRTSimian Immunodeficiency Virus 14Val Gln Arg His Leu Val
Val Glu Thr Gly Thr Thr Glu Thr Met Pro1 5 10 15Lys Thr Ser Arg
201520PRTSimian Immunodeficiency Virus 15Gly Thr Thr Glu Thr Met
Pro Lys Thr Ser Arg Pro Thr Ala Pro Ser1 5 10 15Ser Gly Arg Gly
201620PRTSimian Immunodeficiency Virus 16Thr Ser Arg Pro Thr Ala
Pro Ser Ser Gly Arg Gly Gly Asn Tyr Pro1 5 10 15Val Gln Gln Ile
201719PRTSimian Immunodeficiency Virus 17Ser Gly Arg Gly Gly Asn
Tyr Pro Val Gln Gln Ile Gly Gly Asn Tyr1 5 10 15Val His
Leu1820PRTSimian Immunodeficiency Virus 18Pro Val Gln Gln Ile Gly
Gly Asn Tyr Val His Leu Pro Leu Ser Pro1 5 10 15Arg Thr Leu Asn
201920PRTSimian Immunodeficiency Virus 19Tyr Val His Leu Pro Leu
Ser Pro Arg Thr Leu Asn Ala Trp Val Lys1 5 10 15Leu Ile Glu Glu
202020PRTSimian Immunodeficiency Virus 20Arg Thr Leu Asn Ala Trp
Val Lys Leu Ile Glu Glu Lys Lys Phe Gly1 5 10 15Ala Glu Val Val
202120PRTSimian Immunodeficiency Virus 21Leu Ile Glu Glu Lys Lys
Phe Gly Ala Glu Val Val Pro Gly Phe Gln1 5 10 15Ala Leu Ser Glu
202220PRTSimian Immunodeficiency Virus 22Ala Glu Val Val Pro Gly
Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro1 5 10 15Tyr Asp Ile Asn
202320PRTSimian Immunodeficiency Virus 23Ala Leu Ser Glu Gly Cys
Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn1 5 10 15Cys Val Gly Asp
202420PRTSimian Immunodeficiency Virus 24Tyr Asp Ile Asn Gln Met
Leu Asn Cys Val Gly Asp His Gln Ala Ala1 5 10 15Met Gln Ile Ile
202520PRTSimian Immunodeficiency Virus 25Cys Val Gly Asp His Gln
Ala Ala Met Gln Ile Ile Arg Asp Ile Ile1 5 10 15Asn Glu Glu Ala
202619PRTSimian Immunodeficiency Virus 26Met Gln Ile Ile Arg Asp
Ile Ile Asn Glu Glu Ala Ala Asp Trp Asp1 5 10 15Leu Gln
His2720PRTSimian Immunodeficiency Virus 27Asn Glu Glu Ala Ala Asp
Trp Asp Leu Gln His Pro Gln Pro Ala Pro1 5 10 15Gln Gln Gly Gln
202821PRTSimian Immunodeficiency Virus 28Leu Gln His Pro Gln Pro
Ala Pro Gln Gln Gly Gln Leu Arg Glu Pro1 5 10 15Ser Gly Ser Asp Ile
202920PRTSimian Immunodeficiency Virus 29Gly Gln Leu Arg Glu Pro
Ser Gly Ser Asp Ile Ala Gly Thr Thr Ser1 5 10 15Ser Val Asp Glu
203020PRTSimian Immunodeficiency Virus 30Ser Asp Ile Ala Gly Thr
Thr Ser Ser Val Asp Glu Gln Ile Gln Trp1 5 10 15Met Tyr Arg Gln
203120PRTSimian Immunodeficiency Virus 31Ser Val Asp Glu Gln Ile
Gln Trp Met Tyr Arg Gln Gln Asn Pro Ile1 5 10 15Pro Val Gly Asn
203220PRTSimian Immunodeficiency Virus 32Met Tyr Arg Gln Gln Asn
Pro Ile Pro Val Gly Asn Ile Tyr Arg Arg1 5 10 15Trp Ile Gln Leu
203320PRTSimian Immunodeficiency Virus 33Pro Val Gly Asn Ile Tyr
Arg Arg Trp Ile Gln Leu Gly Leu Gln Lys1 5 10 15Cys Val Arg Met
203420PRTSimian Immunodeficiency Virus 34Trp Ile Gln Leu Gly Leu
Gln Lys Cys Val Arg Met Tyr Asn Pro Thr1 5 10 15Asn Ile Leu Asp
203519PRTSimian Immunodeficiency Virus 35Cys Val Arg Met Tyr Asn
Pro Thr Asn Ile Leu Asp Val Lys Gln Gly1 5 10 15Pro Lys
Glu3620PRTSimian Immunodeficiency Virus 36Thr Asn Ile Leu Asp Val
Lys Gln Gly Pro Lys Glu Pro Phe Gln Ser1 5 10 15Tyr Val Asp Arg
203720PRTSimian Immunodeficiency Virus 37Gly Pro Lys Glu Pro Phe
Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser1 5 10 15Leu Arg Ala Glu
203820PRTSimian Immunodeficiency Virus 38Tyr Val Asp Arg Phe Tyr
Lys Ser Leu Arg Ala Glu Gln Thr Asp Ala1 5 10 15Ala Val Lys Asn
203920PRTSimian Immunodeficiency Virus 39Leu Arg Ala Glu Gln Thr
Asp Ala Ala Val Lys Asn Trp Met Thr Gln1 5 10 15Thr Leu Leu Ile
204020PRTSimian Immunodeficiency Virus 40Ala Val Lys Asn Trp Met
Thr Gln Thr Leu Leu Ile Gln Asn Ala Asn1 5 10 15Pro Asp Cys Lys
204120PRTSimian Immunodeficiency Virus 41Thr Leu Leu Ile Gln Asn
Ala Asn Pro Asp Cys Lys Leu Val Leu Lys1 5 10 15Gly Leu Gly Val
204220PRTSimian Immunodeficiency Virus 42Pro Asp Cys Lys Leu Val
Leu Lys Gly Leu Gly Val Asn Pro Thr Leu1 5 10 15Glu Glu Met Leu
204320PRTSimian Immunodeficiency Virus 43Gly Leu Gly Val Asn Pro
Thr Leu Glu Glu Met Leu Thr Ala Cys Gln1 5 10 15Gly Val Gly Gly
204420PRTSimian Immunodeficiency Virus 44Glu Glu Met Leu Thr Ala
Cys Gln Gly Val Gly Gly Pro Gly Gln Lys1 5 10 15Ala Arg Leu Met
204520PRTSimian Immunodeficiency Virus 45Gly Val Gly Gly Pro Gly
Gln Lys Ala Arg Leu Met Ala Glu Ala Leu1 5 10 15Lys Glu Ala Leu
204620PRTSimian Immunodeficiency Virus 46Ala Arg Leu Met Ala Glu
Ala Leu Lys Glu Ala Leu Ala Pro Val Pro1 5 10 15Ile Pro Phe Ala
204721PRTSimian Immunodeficiency Virus 47Lys Glu Ala Leu Ala Pro
Val Pro Ile Pro Phe Ala Ala Ala Gln Gln1 5 10 15Arg Gly Pro Arg Lys
204820PRTSimian Immunodeficiency Virus 48Pro Phe Ala Ala Ala Gln
Gln Arg Gly Pro Arg Lys Pro Ile Lys Cys1 5 10 15Trp Asn Cys Gly
204920PRTSimian Immunodeficiency Virus 49Gly Pro Arg Lys Pro Ile
Lys Cys Trp Asn Cys Gly Lys Glu Gly His1 5 10 15Ser Ala Arg Gln
205020PRTSimian Immunodeficiency Virus 50Trp Asn Cys Gly Lys Glu
Gly His Ser Ala Arg Gln Cys Arg Ala Pro1 5 10 15Arg Arg Gln Gly
205120PRTSimian Immunodeficiency Virus 51Ser Ala Arg Gln Cys Arg
Ala Pro Arg Arg Gln Gly Cys Trp Lys Cys1 5 10 15Gly Lys Met Asp
205221PRTSimian Immunodeficiency Virus 52Arg Arg Gln Gly Cys Trp
Lys Cys Gly Lys Met Asp His Val Met Ala1 5 10 15Lys Cys Pro Thr Ala
205320PRTSimian Immunodeficiency Virus 53Lys Met Asp His Val Met
Ala Lys Cys Pro Asp Arg Gln Ala Gly Phe1 5 10 15Leu Gly Leu Gly
205420PRTSimian Immunodeficiency Virus 54Cys Pro Asp Arg Gln Ala
Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys1 5 10 15Lys Pro Arg Asn
205520PRTSimian Immunodeficiency Virus 55Leu Gly Leu Gly Pro Trp
Gly Lys Lys Pro Arg Asn Phe Pro Met Ala1 5 10 15Gln Val His Gln
205618PRTSimian Immunodeficiency Virus 56Lys Pro Arg Asn Phe Pro
Met Ala Gln Val His Gln Gly Leu Met Pro1 5 10 15Thr
Ala5720PRTSimian Immunodeficiency Virus 57Met Ala Gln Val His Gln
Gly Leu Met Pro Thr Ala Pro Pro Glu Asp1 5 10 15Pro Ala Val Asp
205820PRTSimian Immunodeficiency Virus 58Met Pro Thr Ala Pro Pro
Glu Asp Pro Ala Val Asp Leu Leu Lys Asn1 5 10 15Tyr Met Gln Leu
205920PRTSimian Immunodeficiency Virus 59Pro Ala Val Asp Leu Leu
Lys Asn Tyr Met Gln Leu Gly Lys Gln Gln1 5 10 15Arg Glu Lys Gln
206021PRTSimian Immunodeficiency Virus 60Tyr Met Gln Leu Gly Lys
Gln Gln Arg Glu Lys Gln Arg Glu Ser Arg1 5 10 15Glu Lys Pro Tyr Lys
206120PRTSimian Immunodeficiency Virus 61Glu Lys Gln Arg Glu Ser
Arg Glu Lys Pro Tyr Lys Glu Val Thr Glu1 5 10 15Asp Leu Leu His
206221PRTSimian Immunodeficiency Virus 62Lys Pro Tyr Lys Glu Val
Thr Glu Asp Leu Leu His Leu Asn Ser Leu1 5 10 15Phe Gly Gly Asp Gln
2063510PRTSimian Immunodeficiency Virus 63Met Gly Val Arg Asn Ser
Val Leu Ser Gly Lys Lys Ala Asp Glu Leu1 5 10 15Glu Lys Ile Arg Leu
Arg Pro Asn Gly Lys Lys Lys Tyr Met Leu Lys 20 25 30His Val Val Trp
Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu Ala Glu 35 40 45Ser Leu Leu
Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu 50 55 60Ala Pro
Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn65 70 75
80Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys His
85 90 95Thr Glu Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val Glu
Thr 100 105 110Gly Thr Thr Glu Thr Met Pro Lys Thr Ser Arg Pro Thr
Ala Pro Ser 115 120 125Ser Gly Arg Gly Gly Asn Tyr Pro Val Gln Gln
Ile Gly Gly Asn Tyr 130 135 140Val His Leu Pro Leu Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Leu145 150 155 160Ile Glu Glu Lys Lys Phe
Gly Ala Glu Val Val Pro Gly Phe Gln Ala 165 170 175Leu Ser Glu Gly
Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys 180 185 190Val Gly
Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn 195 200
205Glu Glu Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln
210 215 220Gln Gly Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly
Thr Thr225 230 235 240Ser Ser Val Asp Glu Gln Ile Gln Trp Met Tyr
Arg Gln Gln Asn Pro 245 250 255Ile Pro Val Gly Asn Ile Tyr Arg Arg
Trp Ile Gln Leu Gly Leu Gln 260 265 270Lys Cys Val Arg Met Tyr Asn
Pro Thr Asn Ile Leu Asp Val Lys Gln 275 280 285Gly Pro Lys Glu Pro
Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser 290 295 300Leu Arg Ala
Glu Gln Thr Asp Ala Ala Val Lys Asn Trp Met Thr Gln305 310 315
320Thr Leu Leu Ile Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys
325 330 335Gly Leu Gly Val Asn Pro Thr Leu Glu Glu Met Leu Thr Ala
Cys Gln 340 345 350Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met
Ala Glu Ala Leu 355 360 365Lys Glu Ala Leu Ala Pro Val Pro Ile Pro
Phe Ala Ala Ala Gln Gln 370 375 380Arg Gly Pro Arg Lys Pro Ile Lys
Cys Trp Asn Cys Gly Lys Glu Gly385 390 395 400His Ser Ala Arg Gln
Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys 405 410 415Cys Gly Lys
Met Asp His Val Met Ala Lys Cys Pro Asp Arg Gln Ala 420 425 430Gly
Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro 435 440
445Met Ala Gln Val His Gln Gly Leu Met Pro Thr Ala Pro Pro Glu Asp
450 455 460Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly Lys
Gln Gln465 470 475 480Arg Glu Lys Gln Arg Glu Ser Arg Glu Lys Pro
Tyr Lys Glu Val Thr 485 490 495Glu Asp Leu Leu His Leu Asn Ser Leu
Phe Gly Gly Asp Gln 500 505 5106425PRTSimian Immunodeficiency Virus
64Met 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 Tyr 20 256525PRTSimian
Immunodeficiency Virus 65Leu 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 Asn
20 256625PRTSimian Immunodeficiency Virus 66Tyr 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 Arg 20 256725PRTSimian Immunodeficiency Virus 67Asn 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 Asn 20 256825PRTSimian Immunodeficiency
Virus 68Arg 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 Glu 20 256925PRTSimian
Immunodeficiency Virus 69Asn 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 Gln
20 257025PRTSimian Immunodeficiency Virus 70Glu 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 Ser 20 257125PRTSimian Immunodeficiency Virus 71Gln 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 Ile 20 257225PRTSimian Immunodeficiency
Virus 72Ser 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 Trp 20 257324PRTSimian
Immunodeficiency Virus 73Thr Met Arg Cys Asn Lys Ser Glu Thr Asp
Arg Trp Gly Leu Thr Lys1 5 10 15Ser Ile Thr Thr Thr Ala Ser Thr
207425PRTSimian Immunodeficiency Virus 74Trp 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 Val 20 257525PRTSimian Immunodeficiency Virus 75Thr 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 Cys 20 257625PRTSimian Immunodeficiency Virus 76Val 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 Lys 20 257725PRTSimian Immunodeficiency Virus
77Cys 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 Lys 20 257825PRTSimian
Immunodeficiency Virus 78Lys 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 Val
20 257925PRTSimian Immunodeficiency Virus 79Lys 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 Arg 20 258025PRTSimian Immunodeficiency Virus 80Val 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 Gln 20 258125PRTSimian Immunodeficiency
Virus 81Arg 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 Arg 20 258225PRTSimian
Immunodeficiency Virus 82Gln 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 Leu
20 258325PRTSimian Immunodeficiency Virus 83Arg 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 Pro 20 258425PRTSimian Immunodeficiency Virus 84Leu 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 Arg 20 258525PRTSimian Immunodeficiency
Virus 85Pro 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 Phe 20 258625PRTSimian
Immunodeficiency Virus 86Arg 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 Tyr
20 258725PRTSimian Immunodeficiency Virus 87Phe 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 Leu 20 258825PRTSimian Immunodeficiency Virus 88Tyr 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 Arg 20 258925PRTSimian Immunodeficiency
Virus 89Leu 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 Met 20 259025PRTSimian
Immunodeficiency Virus 90Arg 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 Asp
20 259125PRTSimian Immunodeficiency Virus 91Met 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 Lys 20 259225PRTSimian Immunodeficiency Virus 92Asp 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 Ile 20 259325PRTSimian Immunodeficiency
Virus 93Lys 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 Thr 20 259425PRTSimian
Immunodeficiency Virus 94Ile 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 Asp
20 259525PRTSimian Immunodeficiency Virus 95Thr 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 Gly 20 259625PRTSimian Immunodeficiency Virus 96Asp 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 Asn 20 259725PRTSimian Immunodeficiency
Virus 97Gly 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 Pro 20 259825PRTSimian
Immunodeficiency Virus 98Asn 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 His
20 259925PRTSimian Immunodeficiency Virus 99Pro 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 Gly 20 2510025PRTSimian Immunodeficiency Virus 100His
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 Leu 20 2510125PRTSimian
Immunodeficiency Virus 101Gly 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 Asn
20 2510225PRTSimian Immunodeficiency Virus 102Leu 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 Thr 20 2510325PRTSimian Immunodeficiency Virus
103Asn 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 Glu 20 2510425PRTSimian
Immunodeficiency Virus 104Thr 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 Ile
20 2510525PRTSimian Immunodeficiency Virus 105Glu 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 Thr 20 2510625PRTSimian Immunodeficiency Virus
106Ile 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 Val 20 2510725PRTSimian
Immunodeficiency Virus 107Thr 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 Ser
20 2510825PRTSimian Immunodeficiency Virus 108Val 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 Gln 20 2510925PRTSimian Immunodeficiency Virus
109Ser 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 Gln 20 2511025PRTSimian
Immunodeficiency Virus 110Gln 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 Glu
20 2511125PRTSimian Immunodeficiency Virus 111Gln 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 Leu 20 2511225PRTSimian Immunodeficiency Virus
112Glu 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 Lys 20 2511325PRTSimian
Immunodeficiency Virus 113Leu 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 Phe
20 2511425PRTSimian Immunodeficiency Virus 114Lys 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 Asn 20 2511525PRTSimian Immunodeficiency Virus
115Phe 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 Trp 20 2511625PRTSimian
Immunodeficiency Virus 116Asn 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 Glu
20 2511725PRTSimian Immunodeficiency Virus 117Trp 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 Gln 20 2511825PRTSimian Immunodeficiency Virus
118Glu 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 Asn 20 2511925PRTSimian
Immunodeficiency Virus 119Gln 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 Ala
20 2512025PRTSimian Immunodeficiency Virus 120Asn 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 Ile 20 2512125PRTSimian Immunodeficiency Virus
121Ala 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 Tyr 20 2512225PRTSimian
Immunodeficiency Virus 122Ile 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 Tyr
20 2512325PRTSimian Immunodeficiency Virus 123Tyr 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 Gln 20 2512425PRTSimian Immunodeficiency Virus
124Tyr 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 Thr 20 2512525PRTSimian
Immunodeficiency Virus 125Gln 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 Gly
20 2512625PRTSimian Immunodeficiency Virus 126Thr 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 Ile 20 2512725PRTSimian Immunodeficiency Virus
127Gly 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 Thr 20 2512825PRTSimian
Immunodeficiency Virus 128Ile 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 Arg
20 2512925PRTSimian Immunodeficiency Virus 129Thr 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 Leu 20 2513025PRTSimian Immunodeficiency Virus
130Arg 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 Arg 20 2513125PRTSimian
Immunodeficiency Virus 131Leu 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 Tyr
20 2513225PRTSimian Immunodeficiency Virus 132Arg 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 Ala 20 2513325PRTSimian Immunodeficiency Virus
133Tyr 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 Trp 20 2513425PRTSimian
Immunodeficiency Virus 134Ala 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 Ala
20 2513527PRTSimian Immunodeficiency Virus 135Trp 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 Leu 20 251367PRTArtificial
SequenceSynthetic 136Ser Tyr Asn Phe Glu Lys Leu1 5
* * * * *