U.S. patent application number 12/106021 was filed with the patent office on 2009-01-15 for sars vaccine compositions and methods of making and using them.
This patent application is currently assigned to Lipid Sciences, Inc.. Invention is credited to Hassibullah Akeefe, Moiz Kitabwalla.
Application Number | 20090017069 12/106021 |
Document ID | / |
Family ID | 56291050 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017069 |
Kind Code |
A1 |
Akeefe; Hassibullah ; et
al. |
January 15, 2009 |
SARS Vaccine Compositions and Methods of Making and Using Them
Abstract
Described is a composition and method for reducing the
occurrence and severity of infectious diseases, especially
infectious diseases such as SARS, 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 immunogenic modified, partially delipidated viral
particles with reduced infectivity. The present invention provides
delipidated viral vaccine compositions, such as therapeutic vaccine
compositions, comprising these modified, partially delipidated
viral particles with reduced infectivity, 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
or, in case of a therapeutic vaccine, to treat or alleviate
infection against the lipid-containing infections 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: |
Akeefe; Hassibullah; (San
Ramon, CA) ; Kitabwalla; Moiz; (Livermore,
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: |
56291050 |
Appl. No.: |
12/106021 |
Filed: |
April 18, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11401434 |
Apr 10, 2006 |
7439052 |
|
|
12106021 |
|
|
|
|
10873015 |
Jun 21, 2004 |
7407662 |
|
|
11401434 |
|
|
|
|
10601656 |
Jun 20, 2003 |
7407663 |
|
|
10873015 |
|
|
|
|
10311679 |
Dec 18, 2002 |
|
|
|
PCT/IB01/01099 |
Jun 21, 2001 |
|
|
|
10601656 |
|
|
|
|
60925628 |
Apr 20, 2007 |
|
|
|
60670574 |
Apr 11, 2005 |
|
|
|
60669738 |
Apr 8, 2005 |
|
|
|
60533542 |
Dec 31, 2003 |
|
|
|
60542947 |
Feb 9, 2004 |
|
|
|
60390066 |
Jun 20, 2002 |
|
|
|
60491928 |
Aug 1, 2003 |
|
|
|
Current U.S.
Class: |
424/221.1 ;
435/235.1 |
Current CPC
Class: |
A61K 2039/5252 20130101;
C12N 2740/16122 20130101; A61K 2039/5258 20130101; C12N 2740/15022
20130101; C12N 2770/20062 20130101; C12N 2730/10122 20130101; C12N
2730/10022 20130101; C07K 14/005 20130101; A61K 39/12 20130101;
A61K 2039/545 20130101; A61P 31/14 20180101; C12N 7/00 20130101;
C12N 2770/24322 20130101; A61P 11/00 20180101; C12N 2770/20034
20130101; A61P 37/04 20180101; A61K 2039/55566 20130101; A61K
2039/5158 20130101; C12N 2770/20022 20130101; C12N 2730/10162
20130101 |
Class at
Publication: |
424/221.1 ;
435/235.1 |
International
Class: |
A61K 39/215 20060101
A61K039/215; C12N 7/01 20060101 C12N007/01 |
Goverment Interests
GOVERNMENT LICENSE RIGHTS
[0002] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of STTR Grant #1 41 AI060267-01 awarded by NIAID.
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2000 |
AU |
PQ8469 |
Dec 28, 2000 |
AU |
PCT/AU00/01603 |
Claims
1. A composition comprising a modified, partially delipidated viral
particle of a coronavirus, wherein the modified, partially
delipidated viral particle of the coronavirus is immunogenic, is of
reduced infectivity as compared to the coronavirus not subjected to
delipidation, and comprises an envelope with envelope viral and
host proteins and a lower lipid content as compared to an envelope
in the coronavirus not subjected to delipidation.
2. The composition of claim 1, wherein the coronavirus causes
SARS.
3. The composition of claim 1, wherein the modified, partially
delipidated viral particle is produced by exposing the coronavirus
not subjected to delipidation to a process comprising treating the
coronavirus with 0.01% to 10% solvent.
4. The composition of claim 3, wherein the solvent is an ether, a
fluoroether, an alcohol, a surfactant, or a combination
thereof.
5. The composition of claim 4, wherein the ether is diisopropyl
ether and the alcohol is butanol.
6. The composition of claim 4, wherein the fluoroether is
sevoflurane.
7. The composition of claim 4, wherein the surfactant is Triton
X100
8. The composition of claim 4, wherein the ether is diisopropyl
ether and the surfactant is Triton X100.
9. The composition of claim 4, wherein the combination thereof is
an alcohol and an ether, an alcohol and a fluoroether, an alcohol
and a surfactant, an ether and a surfactant, or an alcohol, an
ether and a surfactant.
10. A method of creating a modified, partially delipidated viral
particle of a coronavirus comprising the steps of: receiving a
coronavirus in a fluid, exposing the coronavirus to a delipidation
process, comprising treating the coronavirus with 0.01% to 10%
solvent, wherein the delipidation process decreases the lipid
content of a viral envelope of the coronavirus.
11. The method of claim 10, wherein the solvent is an ether, a
fluoroether, an alcohol, a surfactant, or a combination
thereof.
12. The method of claim 11, wherein the ether is diisopropyl ether
and the alcohol is butanol.
13. The method of claim 11, wherein the fluoroether is
sevoflurane.
14. The method of claim 11, wherein the surfactant is Triton
X100
15. The method of claim 11, wherein the ether is diisopropyl ether
and the surfactant is Triton X100.
16. The method of claim 11, wherein the combination thereof is an
alcohol and an ether, an alcohol and a fluoroether, an alcohol and
a surfactant, an ether and a surfactant, or an alcohol, an ether
and a surfactant.
17. A method of attenuating an infection by an coronavirus in an
animal or a human comprising: removing blood containing the
coronavirus from the animal or the human; obtaining plasma from the
blood, the plasma containing the coronavirus; delipidating the
coronavirus by a process comprising contacting the plasma
containing the coronavirus with a 0.01% to 10%% solvent capable of
extracting lipid from the coronavirus to produce modified,
partially delipidated viral particles of the coronavirus, wherein
the modified, partially delipidated particles are of reduced
infectivity and reduced lipid content as compared to the
coronavirus not subjected to the delipidation process, and, wherein
the modified, partially delipidated particles comprise a modified
viral envelope with envelope viral and host proteins, wherein the
contacting is for a time and under conditions sufficient to reduce
the infectivity and the lipid content of the coronavirus to produce
the modified, partially delipidated coronavirus viral particles;
separating the solvent from the modified, partially delipidated
viral particles; and administering the modified, partially
delipidated viral particles of the coronavirus to the animal or the
human in an amount sufficient to produce a cellular immune response
or an antibody response to the coronavirus in the animal or the
human.
18. The method of claim 17, wherein the wherein the solvent is an
ether, a fluoroether, an alcohol, a surfactant, or a combination
thereof.
19. The method of claim 18, wherein the combination thereof is an
alcohol and an ether, an alcohol and a fluoroether, an alcohol and
a surfactant, an ether and a surfactant, or an alcohol, an ether
and a surfactant.
20. A vaccine composition comprising a composition comprising a
pharmaceutically acceptable carrier and a modified, partially
delipidated viral particle of a coronavirus, wherein the modified,
partially delipidated viral particle of the coronavirus is
immunogenic, is of reduced infectivity as compared to the
coronavirus not subjected to delipidation, and comprises an
envelope with envelope viral and host proteins and a lower lipid
content as compared to an envelope in the coronavirus not subjected
to delipidation.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
non-provisional patent application Ser. No. 11/401,434 filed Apr.
10, 2006 which claims the benefit of U.S. provisional patent
application Ser. No. 60/670,574, filed Apr. 11, 2005, U.S.
provisional patent application Ser. No. 60/669,738, filed Apr. 8,
2005, and is a continuation-in-part of U.S. non-provisional patent
application Ser. No. 10/873,015, filed Jun. 21, 2004, which 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, abandoned, which is a U.S.
national phase from PCT patent application number PCT/IB01/01099
filed Jun. 21, 2001, which claims the benefit of Australian patent
application PQ8469 filed Jun. 29, 2000 and PCT patent application
number PCT/AU00/01603 filed Dec. 28, 2000. The present application
also claims the benefit of U.S. provisional patent application Ser.
No. 60/925,628 filed Apr. 20, 2007. 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. U.S. non-provisional patent application Ser. No. 10/873,015,
filed Jun. 21, 2004, 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.
All of these applications are herein incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0003] 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. In a preferred
embodiment, the virus is severe acute respiratory syndrome (SARS)
caused by Coronaviruses. 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
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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, severe acute respiratory syndrome (SARS) caused by
Coronaviruses, meningitis, cytomegalovirus, and hepatitis in its
various forms.
Current Methods of Treatment
[0011] 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.
[0012] 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.
[0013] The current methods of vaccination do have drawbacks, making
them less than optimally desirable for immunizing individuals
against particular pathogens, such as coronavirus and 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, coronavirus,
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. Although antigenic variation has been addressed
via the attempted use of combination drugs or antigens, no prior
art vaccine has succeeded adequately in addressing infections such
as SARS.
[0014] 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)
[0015] Described in the prior art are methods of treating viral
particles with organic solvents and high temperatures thus
dissolving the lipid envelopes and subsequently inactivating the
virus. In those methods, blood is withdrawn from the patient and
separated into two phases--the first phase including red cells and
platelets and the second phase containing plasma, white cells, and
cell-free virus (virion). The second phase is treated with an
organic solvent, thereby killing the infected cells and virions,
and subsequently reintroduced into the patient. In addition to
dissolving the lipid envelope of the virus, the high organic
solvent concentrations cause cell death and damage to the antigens.
Essentially, this method results in a "chemical kill" of the
cell.
[0016] Glutaraldehyde is one such solvent whereby cell inactivation
is achieved as known by those of ordinary skill in the art by
fixation with a dilute solution of glutaraldehyde at about 1:250.
Although treating the virus with glutaraldehyde effectively
delipidates the virus, it also destroys the core. Destruction of
the core is not desirable for producing a modified viral particle
useful for inducing an immune response in a recipient.
[0017] Chloroform is another such solvent. Chloroform, however,
denatures many plasma proteins and is not suitable for use with
biological fluids, which will be reintroduced into the animal or
human. These plasma proteins deleteriously affected by chloroform
serve important biological functions including coagulation,
hormonal response, and immune response. These functions are
essential to life and thus damage to these proteins may have an
adverse effect on a patient's health, possibly leading to
death.
[0018] Further, many of the methods described in the prior art
involve extensive exposure to elevated temperature in order to kill
free virus and infected cells. Elevated temperatures have
deleterious effects on the proteins contained in biological fluids,
such as plasma.
Current Methods of Manufacturing Vaccines
[0019] To date, several manufacturing methods have been employed in
search 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.
[0020] In live attenuated vaccines, the viruses are rendered less
pathogenic to the host, either by specific genetic manipulation of
the virus genome or by passage in some type of tissue culture
system. In order to achieve genetic manipulation, an inessential
gene is deleted or one or more essential genes in the virus are
partially damaged. Upon genetic manipulation, the viral particles
become less virulent yet retain antigenic features. Live attenuated
vaccines can also be used as "vaccine vectors" for other genes,
wherein they act as carriers of genes from a second virus (or other
pathogen) against which protection is required. Attenuated vaccines
(less infective and not inactivated), however, pose several
problems. First, it is difficult to ascertain when the attenuated
vaccine is no longer pathogenic. The risk of viral infection from
the vaccine is too great to properly test for effective
attenuation. In addition, attenuated vaccines carry the risk of
reverting into a virulent form of the pathogen.
[0021] Whole inactivated vaccines are known in the art for
immunizing against infection by introducing killed or inactivated
viruses to introduce pathogen proteins to an individual's immune
system. The administration of killed or inactivated 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.
[0022] Subunit vaccines are yet another form of vaccination well
known to one of ordinary skill in the art. These consist of one or
more isolated proteins derived from the pathogen. These proteins
act as target antigens against which an immune response is
exhibited. The proteins selected for the subunit vaccine are
displayed by the pathogen so that upon infection of an individual
by the pathogen, the individual's immune system recognizes the
pathogen and instigates an immune response. Subunit vaccines are
not whole infective agents and are therefore incapable of becoming
infective.
[0023] DNA vaccine is another type known in the art and uses actual
genetic material of pathogens. In addition, synthetic peptide
vaccines are made up of parts of synthetic peptides. These
synthetic peptides comprise portions of viral proteins chosen
specifically to achieve an anti-viral 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.
[0024] 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.
[0025] What is also needed is an effective means to immunize
individuals against viral pathogen infection that is unique to the
individual due to viral mutations. Preferably the means would
elicit a broad protective immune response with minimized risk of
infecting the individual.
SUMMARY OF THE INVENTION
[0026] The present invention solves the problems described above by
providing a simple, effective and efficient method for treating and
preventing viral infection. In a preferred embodiment, the present
invention provides a simple, effective and efficient method for
treating and preventing SARS 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.
[0027] The present invention is also effective in producing an
autologous, patient-specific therapeutic vaccine against the virus,
by treating a biological fluid containing the virus such that the
virus is present in a modified form, 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.
[0028] 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.
[0029] 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 clade of the coronavirus that causes SARS. 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.
[0030] 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, SARS, 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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. In one embodiment the
vaccine protects against SARS caused by coronavirus.
[0038] 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. In one embodiment, the
infectious viral particle is coronavirus.
[0039] 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. In one embodiment, the positive immune response is to
the coronavirus which causes SARS.
[0040] 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. In one embodiment the viral infection that
is treated is SARS caused by coronavirus.
[0041] 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. In
one embodiment the viral infection that is treated is SARS caused
by coronavirus.
[0042] 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.
[0043] 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.
[0044] The method of the present invention may be used to treat
viruses containing lipid in the viral envelope. A preferred virus
treated with the method of the present invention is the coronavirus
that causes SARS, and subtypes and clades thereof. Other viruses
that can 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.
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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] It is further an object of the present invention to provide
a method for treatment of lipid-containing viruses within a fluid,
which minimizes deleterious effects on proteins contained within
the fluid, thereby creating a modified viral particle with
properties that are capable of initiating a positive immune
response in a patient.
[0052] It is a further object of the present invention to provide a
method for treatment of lipid-containing viruses within a fluid,
which minimizes deleterious effects on proteins contained within
the fluid, thereby creating a modified viral particle with
patient-specific viral antigens.
[0053] 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.
[0054] Another object of the present invention is to completely or
partially delipidate viral particles, wherein the viral particles
comprise coronavirus, immunodeficiency virus, hepatitis in its
various forms, or any other lipid-containing virus, thereby
creating a modified viral particle.
[0055] It is a further object of the present invention to
completely or partially delipidate viral particles, wherein the
viral particles comprise coronavirus, immunodeficiency virus,
hepatitis in its various forms, or any other lipid-containing
virus, while retaining the structural protein core of the
virus.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] It is another object of the present invention to provide a
SARS anti-viral vaccine.
[0060] Another object is to provide a method of modifying viral
particles to prepare a preventative vaccine for SARS.
[0061] Another 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.
[0062] It is a further object of the present invention to lessen
the severity of a disease, particularly SARS, 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.
[0063] 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.
[0064] 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
[0065] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate preferred embodiments
of the present invention.
[0066] FIG. 1 is a Western blot showing MHV antigenicity following
the delipidation procedure with various solvent conditions, as
indicated.
[0067] FIG. 2 shows Western Blots of SARS Spike and SARS
nucleocapsid (NC) proteins post delipidation with various solvent
conditions, as indicated.
[0068] FIG. 3 representative electron micrographs of
.gamma.-irradiated SARS.
[0069] FIG. 4 illustrates the total IgG antibody titers against
SARS CoV Spike and NC post delipidation with various solvent
conditions, as indicated.
[0070] FIG. 5 illustrates the SARS neutralization titers in sera
from mice vaccinated with SARS delipidated with various solvent
conditions, as indicated.
[0071] FIG. 6 illustrates the Spike Ab titers, comparing the titers
in mice vaccinated with delipidated SARS to those in mice
vaccinated with inactivated SARS.
[0072] FIG. 7 illustrates the NC Ab titers comparing the titers in
mice vaccinated with delipidated SARS to those in mice vaccinated
with inactivated SARS.
[0073] FIG. 8 illustrates the neutralization titers comparing the
titers in mice vaccinated with delipidated SARS to mice vaccinated
with inactivated SARS.
[0074] FIG. 9 shows the SARS peptide pools generated and used for
the ELISPOT assay
[0075] FIG. 10 demonstrates IFN-.gamma. ELISPOT responses to SARS
CoV NC and Spike peptide pools (Responses from a representative
mouse/group).
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, particularly coronavirus. 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, fluoroethers (including but not
limited to fluoromethyl hexafluoroisopropyl ether (Sevoflurane)),
surfactants, detergents, and combinations thereof. First extraction
solvents may be combinations such as the following: 1) an alcohol
and an ether; 2) an alcohol and a fluoroether; 3) an alcohol and a
surfactant, 4) an ether and a surfactant; or 5) an alcohol, an
ether and a surfactant. First extraction solvents include, but are
not limited to n-butanol, di-isopropyl ether (DIPE), fluoroether
such as sevoflurane, surfactants such as Triton X-100 or Tween 20,
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,
particularly a coronavirus 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. The modified viral particle has a lower lipid content in
the envelope, displays modified proteins, reduced infectivity and
is immunogenic. Several embodiments of the delipidation methods
provided herein do not lead to destruction of the viral envelope of
the modified, partially delipidated immunogenic viral particles. A
significant proportion of the viral envelopes are present following
the partial delipidation. Thus, some embodiments of the partial
delipidation methods provided herein result in partially
delipidated particles comprising viral envelopes, including
envelope proteins.
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 virus)
and/or from multiple strains or clades of a virus (e.g., SARS virus
or HIV-1 and HIV-2).
[0086] Modified, partially delipidated viral particles obtained
with some embodiments of the methods disclosed herein represent, in
some aspects, new therapeutic vaccine compositions for therapeutic
immunization and induction of an immune response in animals or
humans. In one aspect, modified, partially delipidated viral
particles obtained with the methods disclosed herein are useful for
therapeutic immunization and induction of an immune response in
animals or humans infected by a coronavirus. In one embodiment of
the present invention, administration of the modified, partially
delipidated viral particles and compositions comprising such
particles provides a new method of treatment, alleviation, or
attenuation of coronavirus infections, conditions or clinical
symptoms associated with these infections such as those
coronaviruses leading to the condition known as SARS.
[0087] Partially delipidated coronavirus viral particles obtained
according to some of aspects of the present invention possess at
least some structural characteristics that distinguish them from
the conventional delipidated viruses. Such characteristics include,
but are not limited to, the content of viral proteins, including
viral envelope proteins or host viral membrane associated proteins,
the cholesterol content of the partially delipidated viral
particles, or the ratio of cholesterol content to viral protein.
For example, a partially delipidated coronavirus viral particle
according to some embodiments of the present invention has a lower
cholesterol content than the cholesterol content of the
non-delipidated coronavirus viral particle. In one embodiment, the
lower cholesterol content of the partially delipidated coronavirus
viral particle can be at least 20% to 30% lower than the
cholesterol content of the non-delipidated coronavirus viral
particle. In other embodiments, the cholesterol content in the
modified, partially delipidated coronavirus viral particle is
reduced, for example, no more than 80%, 60%, 55%, or 50% as
compared to the unmodified viral particle. In other embodiments,
the protein content in the modified, partially delipidated
coronavirus viral particle is reduced, for example, no more than
5%, 10%, 15%, 20%, 30%, 40%, 50% or 55% as compared to the
unmodified coronavirus viral particle. According to other
embodiments, the modified, partially delipidated coronavirus viral
particle has a ratio of .mu.g of cholesterol relative to .mu.g of
total protein of at least 0.06.
Infectious Organisms Treated with the Present Invention
[0088] 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, Poxviridae, and any
other lipid-containing virus.
[0089] 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
[0090] 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.
Exemplary Solvent Systems for Use in Removal of Lipid from Viruses
and Effective in Maintaining Integrity of the Viral Particle
[0091] 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 of
solvents 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,
fluoroethers, alcohols, phenols, esters, halohydrocarbons,
halocarbons, amines, detergents, surfactants, 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 and a surfactant, such as
Triton X-100. Another solvent comprises ether or combinations of
ethers, either in the form of symmetrical ethers, asymmetrical
ethers or halogenated ethers such as fluoroethers.
[0092] Suitable first extraction solvents include solvents that
extract or dissolve lipid, including but not limited to alcohols,
hydrocarbons, amines, ethers, fluoroethers (including but not
limited to fluoromethyl hexafluoroisopropyl ether (sevoflurane)),
surfactants, detergents, and combinations thereof. First extraction
solvents may be combinations such as the following: 1) an alcohol
and an ether; 2) an alcohol and a fluoroether; 3) an alcohol and a
surfactant, 4) an ether and a surfactant; or 5) an alcohol, an
ether and a surfactant. First extraction solvents include, but are
not limited to n-butanol, di-isopropyl ether (DIPE), fluoroether
such as sevoflurane, surfactants such as Triton X-100 or Tween 20,
diethyl ether, and combinations thereof.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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 (DIPE)). Asymmetrical ethers may also be
employed. Halogenated symmetrical and asymmetrical ethers may also
be employed. Fluoroethers including but not limited to fluoromethyl
hexafluoroisopropyl ether may also be employed. Halogenated
symmetrical and asymmetrical ethers, such as fluoroethers may be
employed alone or in combination with other solvents in different
ratios such as (sevoflurane:DIPE ratios of 0.01 parts sevoflurane
to 99.99 parts DIPE to 60 parts sevoflurane to 40 parts DIPE, with
a specific ratio range of about 10 parts sevoflurane to 90 parts
DIPE to 5 parts sevoflurane to 95 parts DIPE, with a specific ratio
range of about 10 parts sevoflurane to 90 parts DIPE to 50 parts
sevoflurane to 50 parts DIPE, with a specific ratio range of about
20 parts sevoflurane to 80 parts DIPE to 45 parts sevoflurane to 55
parts DIPE, with a specific range of about 25 parts sevoflurane to
75 parts DIPE.
[0100] Low concentrations of solvents, such as ethers, may be
employed to remove lipids when used alone and not in combination
with other solvents. For example, a low concentration range of
solvents, such as ethers includes but is not limited to 0.5% to
30%, 0.01% to 10%, 0.01% to 5%, 0.1% to 5%, 0.01% to 2%, or 0.1% to
2%, or any number within these ranges. Specific concentrations of
solvents, such as ethers, that may be employed include, but are not
limited to the following: 0.1%, 0.625%, 1.0% 1.25%, 2%, 2.5%, 3.0%,
3.5%, 5.0% and 10% or higher. It has been observed that dilute
solutions of solvents, such as 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] 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.
[0106] Surfactants such anionic and nonionic surfactants may also
be employed alone or together with other solvents. Nonionic
surfactants are known to one of ordinary skill in the art and may
include without limitation surfactants known as Triton, for example
Triton X100 (polyoxyethylene octyl phenyl ether), Tweens such as
Tween 20 (PEG(20)sorbitan monolaurate), or Pluronic (block
copolymers based on ethylene oxide and propylene oxide). When
employed alone, or together with other solvents such as ethers or
lower order alcohols, for example DIPE or n-butanol or combinations
thereof, surfactants may be used in concentrations of from 0.001%
to 1%, 0.07% to 0.8%, 0.05% to 0.5%, or 0.03% to 0.3%.
Biological Fluids and Treatment Thereof for Reducing Infectivity of
Infectious, Lipid-Containing Organisms
[0107] 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, such as coronavirus viral particles, within the plasma
and to create modified coronavirus 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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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 or 20 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
[0113] 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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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)
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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 SARS, HIV,
hepatitis, and/or cytomegalovirus, from the biological sample.
[0122] 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.
[0123] 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
[0124] 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 SARS and HIV, SARS and
influenza, and SARS 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
[0125] 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.
[0126] The formulations may be presented in unit-dose or multi-dose
containers--for example, sealed ampules and vials--and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. The vaccine may be stored at
temperatures of from about 4.degree. C. to -100.degree. C. The
vaccine may also be stored in a lyophilized state at different
temperatures including room temperature. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets commonly used by one of ordinary skill in the
art. The vaccine may be sterilized through conventional means known
to one of ordinary skill in the art. Such means include, but are
not limited to filtration, radiation and heat. The vaccine of the
present invention may also be combined with bacteriostatic agents,
such as thimerosal, to inhibit bacterial growth.
[0127] 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.
[0128] 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.
[0129] 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
[0130] 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
[0131] 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
[0132] 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.
[0133] 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
Development of a Modified Coronavirus Viral Particle for Use as a
Vaccine
[0134] Solvent treatment technology was used to develop a modified
coronavirus viral particle to use as a prophylactic vaccine against
the SARS virus. In addition, solvent-treated virus that was
subsequently subjected to chemical inactivation was tested for the
ability to raise neutralizing antibodies and produce a cellular
immune response in mice. In the following text and elsewhere in the
application, the coronavirus that produces SARS is also referred to
as SARS.
[0135] The SARS stocks used in the experiments were propagated at
the Lovelace Respiratory Research Institute (LRRI), Albuquerque, N.
Mex., in the laboratory of Dr. Kevin Harrod, Director of the
Infectious Disease Program. The initial SARS seed stock was
provided by the Centers for Disease Control (CDC). Supernatants
from SARS infected VERO cells were then sent to Dr. Erdman at the
CDC for inactivation by gamma irradiation.
[0136] The delipidation process was optimized using Mouse Hepatitis
Virus (MHV), provided by Dr. Katherine Holmes at the University of
Colorado-Health Sciences Center. Dr. Holmes provided a seed stock
of MHV as well as the permissive cell line MHV-A59.
[0137] Seven different delipidation methods were developed using
MHV. The methods were characterized based on the amount of
cholesterol removed (measured by the Amplex Red Cholesterol Assay),
and protein recovery. Three of these methods were selected for use
in in vivo mouse immunization studies using gamma irradiated SARS.
Dr. Erdman at the Center for Disease Control gamma irradiated a
stock of SARS with 5.times.10.sup.6 rads, and certified its
inactivation. 1) 3% diisopropyl ether (DIPE) with end-over-end
mixing 20 min at room temperature resulted in 80% cholesterol
removal, and 85% SARS nucleocapsid (NC) recovery, 2) DIPE/Butanol
(n-BuOH) (75:25) at a ratio of 99:1 SARS to solvent (vol:vol) with
end-over-end mixing for 20 min at room temperature resulted in 44%
cholesterol removal and 80% NC recovery, 3) DIPE/Triton X-100
(3%/0.05%) end-over-end mixing for 20 min at room temperature
resulted in 60% cholesterol removal and 85% NC recovery.
[0138] Western blot analysis of the delipidated SARS from each of
the three procedures confirmed the presence of SARS NC and SARS
envelope specific protein (Spike-S). S protein mediates the
receptor binding and membrane fusion process mediated by S protein,
indicating that major viral proteins are present after the
delipidation procedure. (Gallagher, T. M. & Buchmeier, M. J.
(2001) Virology 279: 371-374). This data was similar to the initial
optimization data obtained from delipidating MHV.
[0139] In summary, three unique delipidation processes optimized
for inactivated SARS were developed. Gamma-irradiated SARS was used
for safety reasons and because gamma irradiation was known not to
damage the viral structure of SARS or its antigenicity. Inactivated
SARS was structurally similar to live SARS as visualized by
ultrastructural analysis (kindly preformed by Dr. Humphrey at the
CDC), and by previous findings that gamma irradiation did not
affect the structural integrity or antigenicity of SARS, since
gamma irradiated VERO-E6 cells that were infected with SARS could
be used for immunofluorescence assays (Ksiazek T G et al., 2003 N.
Engl. J. Med. 348:1953-1966). Thus, inactivated SARS was used to
test the utililty of delipidation in enhancing immunogenicity of
SARS vaccine. To determine the immunogenicity of the delipidated
SARS in vivo, a series of in vivo mouse experiments were
performed.
Experiment #1: Evaluation of Delipidation Methods for SARS
[0140] The experiments tested the effects of different delipidation
methods on the ability of the delipidated and inactivated SARS to
generate an immune response. The experiment was designed with four
mice per group. Three groups were tested: [0141] a) Inactivated
purified SARS treated with 3% DIPE; [0142] b) Inactivated purified
SARS treated with DIPE/n-BuOH; [0143] c) Inactivated purified SARS
treated with DIPE/Triton X-100.
[0144] Mice were vaccinated subcutaneously (sc) with 50 ug of
inactivated purified delipidated SARS in incomplete Freund's
Adjuvant in a 50 ul volume in one footpad. SARS was propagated at
Lovelace Respiratory Research Institute (LRRI) by Dr. Kevin Harrod,
and virions were purified by Lipid Sciences, Inc.). At three weeks
post vaccination, mice were sacrificed, and serum was
harvested.
[0145] Serum aliquots were analyzed by Dr. Michael W. Cho at Case
Western Reserve University, where SARS neutralization titers were
established. Dr. Cho's laboratory has established a novel and very
reliable neutralization assay for SARS using pseudotyped murine
leukemia virus (MuLV) with the Spike protein of SARS-coronavirus
(CoV) (or vesicular stomatitis virus-G protein (VSV-G) as a
negative control) as previously described (Han et al., 2004
Virology. 326:140-149). SARS NC and Spike antibody titers were
performed at Lipid Sciences, Inc. using recombinant SARS NC and
Spike purchased from Virolabs, Inc (NJ).
Results: SARS-CoV-specific neutralizing activities were detected in
all three groups above, although the antibody levels were low,
possibly because mice only received one vaccination. Of the three
groups, however, mice vaccinated with SARS delipidated with the
DIPE/Triton X-100 method exhibited slightly higher neutralization
of >50% compared to the other two groups which averaged about
40%. Antibody titers for SARS Spike Protein were similar in all
three groups, while the SARS NC titers were higher in mice
vaccinated with SARS delipidated with 3% DIPE. Therefore, the
"optimal" delipidation was chosen to be the DIPE/Triton X-100
method.
Experiment #2: Dose Escalation Study of Optimally Delipidated SARS
Vaccine
[0146] This experiment tested three different concentrations of
delipidated inactivated purified SARS in comparison to inactivated
purified SARS only, at 0.1 ug, 1 ug, and 10 ug boosts, in a
prime-boost vaccine model, with the aim of enhancing the humoral
immune response observed in Experiment #1. Mice were primed with
SARS supernatant obtained from LRRI in Incomplete Freunds Adjuvant
sc with 100 ug total protein. Two weeks later, mice were boosted
with the appropriate concentration of DIPE:Triton X-100 delipidated
SARS, or inactivated SARS. Four weeks after the boost, mice were
sacrificed and serum collected and sent to Dr. Cho for
determination of neutralizing antibody titers. Serum IgG titers to
SARS NC and Spike were performed at Lipid Sciences, Inc.
Results: Both the inactivated virus and the delipidated inactivated
virus were able to boost neutralizing activity. There were no
significant differences in either the neutralizing antibody titers
or anti-Spike/NC antibody titers, between mice boosted with
DIPE:Triton X-100 delipidated, inactivated SARS virus and mice
boosted with inactivated SARS virus.
[0147] These two studies established that the delipidation process
did not result in major structural damage to the virus.
Immunogenicity was maintained in the delipidated inactivated
virions, since the neutralizing antibody titers were not affected
by the delipidation. Based upon these results, a 10 ug dose was
tested as the boost concentration in Experiment #3.
Experiment #3: Determining the Cell-Mediated Immune Responses in
Mice Boosted with Optimally Delipidated Vaccines
[0148] The experiment tested the cell-mediated immune responses
generated by vaccination with delipidated, inactivated SARS at a
concentration of 10.0 ug. The experiment was designed with four
mice per group in the following four groups: [0149] 1) Boost with
inactivated SARS that was treated with DIPE/Triton X-100; [0150] 2)
Boost with inactivated SARS; [0151] 3) Primed only; and [0152] 4)
No prime/no boost.
[0153] All mice in groups 1, 2, and 3 were primed with the same
SARS supernatant obtained from LRRI, as per Experiment #2. Mice
were injected two weeks post priming, and sacrificed one week after
the booster injection since cell mediated immune responses in mice
peak at about one week after the booster injection. Cell-mediated
immune responses were measured using murine interferon-gamma
(IFN-.gamma.) ELISPOT Assay, per manufacturer's protocol (MABTech).
Cells were incubated with pools of peptides covering the entire
SARS CoV NC and Spike proteins.
[0154] The following reagent was obtained through the NIH
Biodefense and Emerging Infections Research Resources Repository,
NIAID, NIH: SARS Overlapping Peptide Array, NR-143. Pools of
peptides (eight peptides per pool, 13- to 20-mers with
approximately 10 amino acid overlaps) were generated and used in
the enzyme-linked immunosorbent spot (ELISPOT Assay: Czerkinsky C,
et al., (1983) "A solid-phase enzyme-linked immunospot (ELISPOT)
assay for enumeration of specific antibody-secreting cells". J
Immunol Methods 65 (1-2): 109-21.).
Results: Mice boosted with delipidated, inactivated SARS had
enhanced immune responses to Spike and NC peptide pools compared to
booster injection with inactivated SARS. There was a boosting
effect observed, compared to the no-boost Group. Booster injections
with delipidated, inactivated SARS enhanced cell-mediated immune
response, but did not change the antibody titers of Spike and NC.
Conclusion: The above findings indicate that
delipidated/inactivated SARS generated better cell mediated immune
responses than a preparation of inactivated SARS alone. The
enhanced cell-mediated immune responses may greatly assist in
preventing establishment of infection due to the broad epitope
recognition in recipients primed with our vaccine.
[0155] Production of mouse neutralizing antibodies was measured
with a neutralization assay for SARS using pseudotyped murine
leukemia virus (MuLV) expressing the Spike protein of SARS-CoV (or
VSV-G as a negative control) as previously described (Han et al.,
2004 Virology. 326:140-149). Cell mediated immune responses were
measured using ELISPOT Assays for IFN-.gamma.. ELISPOT Assays have
been used extensively for measuring cell mediated immune responses
in several different disease models.
[0156] The experiments demonstrated the utility of the delipidation
process in creating a SARS vaccine: 1) capable of triggering a
strong cell-mediated immune response; 2) of increased efficacy; and
3) usable separately or as a part of a component vaccine. In
addition, this delipidation process is easy to perform and easily
scaled for commercial production.
Experimental Protocols: Optimization of Solvent and Chemical
Treatment for SARS Coronavirus and Evaluation of Native Viral
Protein Structure and Viral Envelope Changes Post Treatment
MHV & SARS Viral Purification
MHV Growth and Purification:
[0157] MHV-A59, the MHV permissive cell line 17CL.1, and A04 (a
polyclonal goat anti-MHV antibody), were kindly provided by Dr.
Kathryn Holmes, University of Colorado Health Sciences. MHV was
propagated in 17CL.1 and purified according to Sturman, et al. (J.
Virol 1980 33:449-462). Briefly, viral supernatant was precipitated
using polyethylene glycol (PEG, Sigma, St. Louis, Mo.) at a final
concentration of 10%, incubated for 15 min at room temperature
(RT), and pelleted at 10,000.times.g for 1 hr at 4.degree. C. The
pellet was resuspended in 4 ml of tris-maleate buffer (TME, Sigma,
St. Louis, Mo.), pH 6.0, and layered on top of the 20%-55% sucrose
gradient layer. Virus was pelleted by spinning at 32,000 rpm for 4
hr at 4.degree. C. in a 80 Ti rotor (Beckman Coulter, Fullerton,
Calif.). Aliquots (0.4 ml) were collected from the bottom of the
tube, and quantitated using the Biorad Total Protein Assay (Biorad,
Hercules, Calif.).
SARS Growth and Purification:
[0158] SARS CoV-Utah strain was obtained from the Centers for
Disease Control (CDC). SARS permissive cell line VERO-E6 stock was
obtained from ATCC Inc. SARS was propagated in VERO-E6 cells in the
laboratory of Dr. Kevin Harrod, Lovelace Respiratory Research
Institute (LRRI), Albuquerque, N. Mex., and purified using a
modified MHV purification protocol, in which SARS supernatant was
not PEG precipitated. SARS supernatant was directly layered on top
of a 20%-55% sucrose gradient layer, and pelleted at 32,000 rpm for
4 hr at 4.degree. C. in a 80 Ti rotor (Beckman Coulter, Fullerton,
Calif.). Aliquots (0.4 ml) were collected from the bottom of the
tube, and analyzed using both the SARS Ag ELISA kit (MedQuick
Testing, SimiValley, Calif.) and the Biorad Total Protein Assay kit
(Biorad, Hercules, Calif.).
SARS ELISA:
[0159] To quantify the amount of SARS antigen, a SARS-CoV-Ag ELISA
kit (MedQuick Testing, SimiValley, Calif.) targeting SARS NC
protein, was used according to the manufacturer's instructions.
Cholesterol Assay:
[0160] Cholesterol in the viral fractions was analyzed by the
Amplex Red Total Cholesterol Assay according to manufacturer's
protocol (Molecular Probes, Eugene, Oreg.).
Western Blot:
[0161] MHV Western Blot:
[0162] For both Western blot and Coomasie staining, 10% SDS-PAGE
gels (Biorad, Hercules, Calif.) were used. Samples were loaded at 7
ul (concentration of 2 mg/ml) and 7 ul of 2.times. loading dye with
2-mercaptoethanol (2-ME, Sigma, St. Louis, Calif.). The gels were
run at 220 V constant voltage for 1 hr. The Western blot gel was
transferred to a nitrocellulose membrane (Biorad, Hercules,
Calif.), and blocked with 5% milk and 0.1% Tween-20 (Sigma, St.
Louis, Mo.) in a tris-glycine buffer (Biorad, Hercules, Calif.).
The primary antibody AO4 (goat anti-MHV Ab) was used at 1:2000
dilution. The secondary antibody was a rabbit anti-goat horseradish
peroxidase (HRP) (Sigma, St. Louis, Mo.) used at 1:5000 dilution. A
colorimetric substrate diaminobenzidine (DAB) Enhanced Liquid
Substrate System for Membrane ELISA, Sigma, St. Louis, Mo.) for HRP
was used for Western blot development.
[0163] SARS Western Blot:
[0164] For both Western blot and Coomasie staining, 10% SDS-PAGE
gels (Biorad, Hercules, Calif.) were used. Samples were loaded at 7
ul (concentration of 2 mg/ml) and 7 ul of 2.times. loading dye with
2-mercaptoethanol (2-ME, Sigma, St. Louis, Mo.). The gels were run
at 220 V constant voltage for 1 hr. The Western blot gel was
transferred to a nitrocellulose membrane (Biorad, Hercules,
Calif.), and blocked with 5% milk and 0.1% Tween-20 (Sigma, St.
Louis, Mo.) in a tris-glycine buffer (Biorad, Hercules, Calif.).
The primary antibody solution was a cocktail of mouse monoclonal
antibodies against SARS Spike and NC (Imgenex, San Diego, Calif.).
Positive controls for Spike and NC (Virolabs, Chantilly, Va.) were
run at 3.5 ug each. The secondary antibody, a goat anti-mouse-HRP
polyclonal antibody (Sigma, St. Louis, Mo.) was used at a dilution
of 1:5000. A chemiluminescent substrate (ECL+, Amersham
BioSciences, Piscataway, N.J.) for HRP was used for Western blot
development. The membrane was developed on a Kodak MS Film (Eastman
Kodak, Rochester, N.Y.).
MHV Delipidation Procedures:
[0165] A total of seven different delipidation matrices were
developed, using MHV as a surrogate for SARS, due to SARS material
constraints:
1. Diisopropylether (DIPE, VWR, West Chester, Pa.) 17 ul/ml,
end-over-end (EOE) mixing for 20 min at room temperature (RT).
Solvent was removed by passing the mixture through an activated
charcoal column. 2. DIPE and n-butanol (n-BuOH, VWR, West Chester,
Pa.) at a 95:5 mixture and at a 99:1 virus:solvent ratio (vol:vol),
20 min EOE mixing at RT. Solvent was removed by passing the mixture
through an activated charcoal column. 3. DIPE and n-butanol
(n-BuOH) at a 95:5 mixture and at a 99:1 virus:solvent ratio
(vol:vol), vortexed at high speed for 15 sec at RT. Solvent was
removed by passing the mixture through an activated charcoal
column. 4. BuOH and sevoflurane (Abbott Labs, Abbott Park, Ill.) at
a 75:25 mixture and at a 1:2 virus:solvent ratio (vol:vol),
vortexed high speed for 15 sec, with gravity separation at RT. The
aqueous layer was removed. Solvent was removed by passing the
mixture through an activated charcoal column. 5. A final
concentration of 3% DIPE and 0.05% Triton X-100, EOE mixing for 20
min at RT. Solvent was removed by passing the mixture through an
activated charcoal column. 6. A final concentration of 3% DIPE, EOE
mixing for 20 min at RT. Solvent was removed by passing the mixture
through an activated charcoal column. 7. A final concentration of
0.05% Triton X-100 (Sigma, St. Louis, Mo.), EOE mixing for 20 min
at RT. Solvent was removed by passing the mixture through an
activated charcoal column.
SARS Delipidation Procedure:
[0166] The three delipidation procedures selected for SARS
delipidation experiments were:
1. DIPE and n-butanol (n-BuOH) at a 95:5 mixture and at a 99:1
virus:solvent ratio (vol:vol), 20 min EOE with mixing at RT.
Solvent was removed by passing the mixture through an activated
charcoal column. 2. A final concentration of 3% DIPE and 0.05%
Triton X-100, EOE mixing for 20 min at RT. Solvent was removed by
passing the mixture through an activated charcoal column. 3. A
final concentration of 3% DIPE, EOE mixing for 20 min at RT.
Solvent was removed by passing the mixture through an activated
charcoal column.
Electron Microscopy:
[0167] Electron micrographs (EMs) of .gamma.-irradiated SARS were
made by Dr. Dean Erdman and Dr. Charles Humphrey at the CDC. SARS
supernatants containing about 1.times.10.sup.6 PFU/ml were gamma
irradiated with 5.times.10.sup.6 rads, and certified inactivated by
Dr. Dean Erdman.
Results
TABLE-US-00001 [0168] TABLE 1 Optimization of delipidation protocol
using MHV as a surrogate for SARS Percent Percent Protein
Cholesterol Solvent Recovered Removed 1 DIPE 80.26 39.20 2
DIPE:n-Butanol, EOE 91.43 43 3 DIPE:n-Butanol, vortex 85.1 31 4
n-Butanol:Sevoflurane 48.32 ND* below detection level 5 DIPE:Triton
X-100 70.95 70.35 6 DIPE (3%) 75.45 70 7 Triton X-100 78.02 25.5
(0.05%)
[0169] Table 1 summarizes the data obtained from the seven
delipidations performed on MHV, a surrogate for SARS. Protein
recoveries post-delipidation were >70%, except for
n-BuOH:Sevoflurane delipidated virus, where the recovery was
48%.
[0170] The following three delipidation protocols were used on
purified SARS virus:
1. DIPE and n-butanol (n-BuOH) at a 95:5 mixture and at a 99:1
virus:solvent ratio (vol:vol), 20 min EOE, RT. Solvent was removed
by passing the mixture through an activated charcoal column. This
is protocol #2 in Table 1. 2. A final concentration of 3% DIPE and
0.05% Triton X-100, EOE 20 min at RT. Solvent was removed by
passing the mixture through an activated charcoal column. This is
protocol #5 in Table 1. 3. A final concentration of 3% DIPE, EOE 20
min at RT. Solvent was removed by passing the mixture through an
activated charcoal column. This is protocol #6 in Table 1.
TABLE-US-00002 TABLE 2 Protein and Cholesterol Results: SARS
delipidation methods Percent Nucleocapsid Percent Cholesterol
Solvent Recovered Removed 1 DIPE (3%) 85 80 2 DIPE:Triton X-100 85
60 3 DIPE:n-Butanol (95:5) 80 44
[0171] Table 2 summarizes the data obtained from the three chosen
delipidation methods performed on SARS. The protein recoveries as
measured by the SARS ELISA detecting SARS NC were all .gtoreq.80%.
The cholesterol removal was similar to those observed in MHV, as
seen in Table 1.
[0172] FIG. 1 illustrates a Western Blot performed on delipidated
MHV as discussed in Table 1. The polyclonal anti-MHV antibody A04
was kindly provided by Dr. Kathryn Holmes. All lanes show positive
reactivity with the anti-MHV Ab. The Western Blot for samples
delipidated by method #1, and #3 of the delipidation matrix in
Table 1 also showed the same patterns of staining as the DIPE:
triton delipidated SARS (data not shown).
[0173] FIG. 2 shows Western Blots of SARS Spike and SARS NC
proteins post delipidation. Lanes 1-3 correspond to samples 1-3 in
the SARS delipidation methods listed in Table 2 above. Lane 4 is
purified SARS Spike and NC proteins from Virolabs. Spike reactivity
is seen in 3% DIPE delipidated SARS, while very strong reactivity
to NC was observed in all three delipidation protocols. The primary
antibodies used in SARS Western blots were monoclonal
antibodies.
[0174] FIG. 3 shows representative electron micrographs of
.gamma.-irradiated SARS The EMs of virus pre- and post-irradiation
were not significantly different. The picture on the right, a
magnification of 23000.times., has a typical SARS CoV appearance.
The Spike proteins are clearly seen on the membrane, and the
morphology is consistent with those of SARS CoV.
Discussion
[0175] By using MHV as a surrogate for SARS, seven different
delipidation protocols were evaluated, as listed in Table 1. The
n-BuOH/sevoflurane method clearly elicited major structural damage,
as inferred by the protein recovery. Western blot analysis of the
various delipidated MHV (FIG. 1) showed that viral proteins were
intact and immunogenic. No significant loss of viral proteins was
seen in any of the delipidation methods, except the
sevoflurane:n-BuOH delipidated samples. Protocols 1, 3, 4, and 7
(Table 1) were eliminated due to the low percentage of cholesterol
removal.
[0176] Three delipidation protocols were tested on purified SARS
CoV as listed in Table 2. Western blot analysis of the various
delipidated SARS samples showed that reactivity to SARS NC was very
strong in all delipidated samples. Viruses delipidated with 3% DIPE
showed readily detectable reactivity to SARS Spike protein, while
the other two samples did not.
[0177] Ultrastructural analyses were performed on irradiated SARS
supernatant to determine the effects of irradiation on the viral
structure. It was confirmed that the cultures were SARS CoV and
that irradiation did not significantly alter the virion structure.
The results indicated development of three unique delipidation
methods for SARS CoV, which were tested for their in vivo
immunogenicity.
Testing the Ability of Solvent and Chemically Treated Virions to
Produce an Immune Response
[0178] The ability of solvent and chemically treated SARS virions
to produce an immune response was examined by:
A. Vaccinating mice with solvent and chemically treated SARS
virions; B. Testing for production of mouse neutralizing antibodies
in serum using Vero E6 cell cytopathic assay; and, C. Evaluating
mouse cellular response to vaccination with solvent-treated SARS
virions.
[0179] The immunogenicity of delipidated SARS in vivo, in a murine
model was tested. Three experimental protocols were used:
1) Evaluation of three Different Delipidation Methods for SARS; 2)
Dose Escalation Study of Optimally Delipidated SARS Vaccine; and,
3) Determining The Cell-Mediated Immune Responses in Mice Boosted
with Optimally Delipidated Vaccines.
Experiment #1 Evaluation of Three Different Delipidation Methods
for SARS
[0180] The particles obtained by three delipidation processes were
tested with respect to generating an immune response. The
experiment was designed with three mice per group testing the
following three groups:
A. Inactivated SARS treated with 3% DIPE; B. Inactivated SARS
treated with DIPE/n-BuOH (95:5); and, C. Inactivated SARS treated
with 3% DIPE/0.05% Triton X-100.
[0181] Mice were vaccinated sc with 50 .mu.g of inactivated
delipidated SARS in 50 .mu.l in one footpad. At three weeks post
vaccination, mice were sacrificed, and serum was harvested.
[0182] Serum aliquots were sent to the laboratory of Dr. Michael W.
Cho (Case Western Reserve University), where SARS neutralization
titers were evaluated using a neutralization assay for SARS using
pseudotyped murine leukemia virus (MuLV) with the Spike protein of
SARS-CoV (or VSV-G as a negative control) as previously described
(Han et al., 2004 Virology. 326:140-149). SARS NC and Spike
antibody titers were performed at Lipid Sciences, Inc. using
recombinant SARS NC and Spike purchased from Virolabs, Inc (NJ)
Materials and Methods
[0183] Pseudotyped SARS Neutralization Assay:
[0184] Neutralization assays were performed using pseudotyped MuLV
with Spike protein of SARS-CoV (or VSV-G as negative control) as
previously described (Han et al., Virology. 326: 140-149). Briefly,
VERO-E6 cells were used for the pseudovirus infection and were
plated at 0.5.times.10.sup.4 cells per well in a 96-well plate one
day before infection. Heat-inactivated plasma samples at indicated
dilutions were incubated with 100 infectious units of
pseudoviruses. The control and experimental samples of each
dilution were then dispensed to the triplicate wells containing the
VERO-E6 cells for 1 hr at 37.degree. C. After removing the
serum:virus mix, cells were further incubated in DMEM with 5% FBS
at 37.degree. C. in a 5% CO.sub.2 incubator for 1.5 days. To
determine the neutralization activity, the Beta-Glo assay system
(Promega, Madison, Wis.) was used according to manufacturer's
protocol. Cells were washed with PBS, and lysed with 100 .mu.l of
Report Lysis Buffer. 75 .mu.l of cell lysates and 75 .mu.l of
Beta-Glo reagent were mixed in a white-walled plate. The mixtures
were incubated for 30 min at RT and measured using a luminometer
(Biorad, Hercules, Calif.).
[0185] SARS Spike and NC Antibody Titers:
[0186] Serum samples were titrated for antibodies to viral epitopes
using routine EIA analysis. Briefly, high protein binding ELISA
micro plates (Fisher, Pittsburgh, Pa.) were incubated with 1 .mu.g
purified recombinant SARS Spike or SARS NC protein (Virolabs,
Chantilly, Va.) overnight in standard bicarbonate coating buffer,
pH 9.6 at 4.degree. C. Following three washes with PBS/Tween 20,
the plates were blocked for 1 hr at RT with PBS containing 5%
normal goat serum (Sigma, St. Louis, Mo.). Serial 1:5 dilutions of
the sera to be tested in PBS containing 5% normal goat serum
starting at 1:500, were added to the wells for 1 hr at RT. After
washing the unbound antibodies, the plates were incubated with an
HRP-anti mouse IgG conjugate at 1:5000 (Sigma, St. Louis, Mo.), and
developed using tetramethylbenzidine (TMB) substrate (Sigma, St.
Louis, Mo.). Plates were read at a 405 nm wavelength using an ELISA
plate reader (Molecular Devices, Sunnyvale, Calif.).
[0187] ELISPOT Assay:
[0188] Mouse interferon-gamma (IFN-.gamma.) ELISPOT assays were
performed using splenocytes to determine the cell-mediated immune
responses generated post-vaccination with delipidated SARS.
Briefly, 96 well Millipore ELLIP 10SSP multiscreen plates
(Millipore, Billerica, Mass.) were coated with 100 .mu.l anti-mouse
IFN-.gamma. capturing antibody (MABTECH, Cincinnati, Ohio,
monoclonal Ab clone AN-18). The capturing Ab was diluted to 10
mg/ml in sterile PBS. Plates were blocked with 150 .mu.l/well of
10% RPMI (RPMI 1640 containing; 10% FBS, 10 mM HEPES buffer, 2 mM
glutamine, 0.5 mg/ml gentamicin, and 50 mM 2-mercaptoethanol) and
the plates incubated at room temperature for at least 2 hours. The
peptide pools mentioned above were added directly to wells in a
volume of 50 .mu.l and then freshly isolated splenocytes were added
at a concentration of 10.sup.5 cells/well in 50 .mu.l of 10% RPMI
media. The final concentration of the peptides in the screening
assay was 10 mM. Plates were incubated for three days at 5%
CO.sub.2 at 37.degree. C. washed and 100 l/well of 2 mg/ml
biotinylated anti-IFN-.gamma. mAb (clone R4-6A2, MABTECH,
Cincinnati, Ohio) in PBS were added and incubated at room
temperature for 3 h, followed by 100 .mu.l/well avidin peroxidase
conjugate (APC) for 1 hr. After washing, ELISPOTs were developed
using the Vectastain ABC Kit (Vector Laboratories, Burlingame,
Calif.) according to manufacturer's protocol. The number of
spots/10.sup.5 cells/well in the ELISPOT plates were read in a
plate reader in the Vanderbilt University Core Facility, Nashville,
Tenn.
Results
[0189] FIG. 4 illustrates the total IgG antibody titers against
SARS CoV Spike and NC. The titers obtained from all three
delipidated virus vaccines were similar for both Spike and NC.
Although mice vaccinated with DIPE:n-BuOH showed slightly higher NC
titers, the differences were not significant. Data reflect mean
O.D. (standard deviation of <10%). IgM specific titers for all
samples were at an O.D. of <1.0 indicating the presence of very
low IgM antigen specific antibodies (data not shown).
[0190] FIG. 5 illustrates the SARS neutralization titers, performed
by Dr. Michael Cho. Sera from mice vaccinated with DIPE:Triton
X-100 treated SARS showed slightly better titers than mice
vaccinated with SARS treated with the other delipidation protocols.
SARS-CoV-specific neutralizing activities were detected in the sera
from all three groups above. The overall antibody levels were low,
possibly because mice only received one vaccination. Of the three
groups, however, mice vaccinated with SARS delipidated with the
DIPE/Triton X-100 method exhibited slightly higher neutralization
of >50% compared to the other two groups which averaged at about
40%.
[0191] The DIPE/Triton X-100 delipidation method was used in
Experiment #2, where we performed a titration of three
concentrations of DIPE:Triton X-100 delipidated SARS vaccine of 0.1
.mu.g, 1 .mu.g, and 10 .mu.g of protein, as discussed below.
Experiment #2 SARS-Dose Escalation Study of Optimally Delipidated
SARS Vaccine
[0192] The objective of this study was to test three different
concentrations of delipidated inactivated purified SARS with
inactivated purified SARS only, at 0.1 .mu.g, 1 .mu.g, and 10 .mu.g
boosts. Mice (3 mice/group) were primed with SARS supernatant
(virus was unpurified) obtained from LRRI that was
.gamma.-irradiated by Dr. Erdman at the CDC and had a protein
concentration of 4 mg/ml. Mice were primed using Incomplete Freunds
Adjuvant sc with 100 .mu.g total protein in a volume of 500 .mu.l.
Two weeks later, mice were boosted with the appropriate
concentration of delipidated SARS, or inactivated SARS in a volume
of 500 .mu.l administered sc. Four weeks after the booster
injection, mice were sacrificed and serum collected and sent to Dr.
Cho for neutralizing antibody titers. Serum IgG titers to SARS NC
and Spike were performed at Lipid Sciences, Inc.
Results
[0193] FIG. 6 illustrates the Spike Ab titers from Experiment #2,
comparing the titers in mice vaccinated with delipidated SARS to
those in mice vaccinated with inactivated SARS. FIG. 7 illustrates
the NC Ab titers comparing the titers in mice vaccinated with
delipidated SARS to those in mice vaccinated with inactivated SARS.
A clear increase in Ab titers was observed for both antigens with
the 10 .mu.g booster injection. The Spike and NC titers in
delipidated SARS boost showed a clear dose-response. The titers
obtained from inactivated SARS boost showed a clear enhancement in
titers following the 10 .mu.g boost. However, the overall patterns
of Ab titers in both groups were surprisingly similar. When
comparing the overall Ab titers to mice primed only, the patterns
were similar as well. IgM specific titers for all samples were at
an O.D. of <1.0 indicating the presence of very low IgM antigen
specific antibodies (data not shown).
[0194] FIG. 8 illustrates the neutralization titers from Experiment
#2 comparing the titers in mice vaccinated with delipidated SARS to
mice vaccinated with inactivated SARS. Titers obtained from mice
vaccinated with 10 .mu.g delipidated SARS had the highest
neutralization titers, followed by mice vaccinated with 1 .mu.g,
then 0.1 .mu.g delipidated SARS (FIG. 8 top panel). The
neutralization titers in mice boosted with either delipidated or
inactivated SARS (FIG. 8 bottom panel) were not significantly
different, although a clear boosting response was observed when
compared to the primed only group. These results were surprising,
since the Ab titers to Spike and NC were similar in all groups
primed and boosted.
[0195] From these results, the dose of 10 .mu.g was chosen as the
booster injection dose in Experiment #3, which evaluated the
cell-mediated immune responses measured using the mouse IFN-.gamma.
ELISPOT assay in vaccinated mice.
Experiment #3 SARS-Determining the Cell-Mediated Immune Responses
in Mice Boosted with Optimally Delipidated Vaccines
[0196] The experiment tested the cell-mediated immune responses
generated by vaccination with DIPE:Triton X-100 delipidated SARS at
a concentration of 10 .mu.g. The experiment was designed with four
mice per group testing the following four Groups: 1) Boosting with
inactivated SARS that was treated with DIPE:Triton X-100; 2)
Boosting with inactivated SARS; 3) Primed only; and, 4) No prime/no
boost.
[0197] All mice in groups 1, 2, and 3 were primed with the same
SARS supernatant (100 .mu.g sc in incomplete Freunds adjuvant
obtained from LRRI), as per Experiment #2. Mice received a booster
injection two weeks post priming i.v., and were sacrificed one week
later since cell mediated immune responses in mice peak at about
one week after a booster injection. Cell-mediated immune responses
were measured using murine interferon-gamma (IFN-.gamma.) ELISPOT
Assay, per manufacturer's protocol (MABTech). Cells were incubated
with pools of peptides covering the entire SARS CoV NC and Spike
proteins (FIG. 9).
[0198] Pools of peptides (eight peptides per pool for NC, 12
peptides per pool for Spike, 13- to 20-mers with approximately 10
amino acid overlaps) were generated using SARS Overlapping Peptide
Array, which was obtained through the NIH Biodefense and Emerging
Infections Research Resources Repository, NIAID, NIH. The four
digit number in each cell of each table represents a peptide whose
amino acid sequence in shown in FIG. 9. The numbers 1-7 at the top
of each column for the top table called NC are pool components. For
example, pool 1 contains peptides 9539-9589 for NC peptides pool.
The amino acid sequences for each NC peptide shown in FIG. 9 is
included in Table 3.
[0199] The numbers 1-12 at the top of each column for the bottom
table called Spike are pool components. For example, pool 12
contains peptides 9609-9765 for the Spike peptide pool. The amino
acid sequences for each S peptide shown in FIG. 9 is included in
Table 4.
[0200] The ELISPOT data showed a significantly enhanced peptide
pool response for both NC and Spike in mice vaccinated with
delipidated SARS, compared to either the inactivated SARS
vaccination, or primed only mice. Surprisingly, mice vaccinated
with inactivated SARS did not show robust ELISPOT responses,
compared to mice boosted with delipidated SARS.
Discussion
[0201] The experiments measured the immune responses in vivo in
mice vaccinated with delipidated or inactivated SARS. Experiment #1
tested delipidation methods for use in the dose-escalation study in
Experiment #2. We focused on a single immunization with SARS
delipidated by 3% DIPE, DIPE:n-BuOH, or DIPE:Triton X-100. Total
IgG Ab titers to SARS Spike and NC antigens were measured, as well
as the neutralization Ab titers. FIG. 4 illustrates the Ab titers
to SARS Spike and NC. The Ab titers from SARS delipidated with the
three different methods were similar. The neutralization Ab titers
(FIG. 5) showed that mice vaccinated with DIPE:Triton X-100 had
slightly higher neutralization titers (>60%). The overall titers
were low compared to the positive control sera (data not shown),
probably because mice were given one vaccination prior to serum
collection.
[0202] Experiment #2 was a prime/boost protocol, which compared
boosting primed mice with 0.1 .mu.g, 1 .mu.g, and 10 .mu.g
inactivated SARS or DIPE:Triton X-100 delipidated SARS vaccines.
Priming was performed using a SARS supernatant propagated at LRRI,
which had been irradiated by Dr. Dean Erdman at the CDC. The
prime/boost strategy was adopted because this protocol would
enhance both cell- and humoral immune responses. Total Ab titers to
SARS Spike and NC were similar in mice boosted with either
inactivated or delipidated SARS (FIG. 6-Spike, FIG. 7-NC). These
titers were higher than in mice that were primed only and did not
receive a booster injection, indicating a clear enhancement of
humoral immune responses by adding a booster injection. A
dose-dependant increase in Ab titers was observed in delipidated
SARS boosted mice; overall, the 10 .mu.g booster injections gave
higher titers than other doses used for booster injections. The
neutralizing Ab titers (FIG. 8) also showed a dose-dependant
response: 10 .mu.g>1 .mu.g>0.1 .mu.g booster injections.
[0203] It was decided that a booster injection of 10 .mu.g
DIPE:Triton X-100 delipidated SARS, and the resulting cell-mediated
immune responses (measured by IFN-.gamma. ELISPOT) would be
compared to a 10 .mu.g inactivated SARS boost. The ELISPOT plates
were read using an ELISPOT plate reader. The ELISPOT data (FIG. 10)
was surprising. Mice boosted with delipidated SARS had greatly
enhanced responses to both Spike and NC peptide pools, compared to
mice boosted with inactivated SARS. These data were surprising,
since the neutralizing Ab titers in the two groups were similar,
and the total Ab titers for both Spike and NC were similar in the
two groups.
[0204] The data indicate that delipidated SARS vaccination clearly
enhanced cell mediated immune responses. The current data indicated
that delipidation may favor augmentation of cell mediated immune
responses by either enhancing existing pathways of antigen
processing and presentation, or by using additional and more
efficient pathways of antigen processing and presentation.
Virus Purification: A Direct Pelleting Procedure for Purifying
SARS
[0205] Gamma-irradiated SARS supernatant was used for priming mice
in the prime/boost Experiment #2 and 3. The SARS supernatant used
to generate the vaccines for Experiment #1-3, yielded highly
purified SARS for the boosting doses, post purification using Dr.
Holmes method (as discussed below). Mouse hepatitis virus (MHV) was
kindly provided by Dr. Kathryn Holmes, at the University of
Colorado Health Sciences. Dr. Holmes purification method for MHV
entailed layering viral supernatant onto a 20-55% sucrose gradient
made in a tris-maleate buffer.
[0206] This purification method for SARS using a different SARS
culture propagated by LRRI, was used to yield highly purified virus
that was subsequently used as the boosts for Experiment #1, 2, and
3. It was determined that SARS can be efficiently purified by
layering the supernatant on a 20-55% sucrose gradient and spinning
at 32,000 rpm for 4 hr at 4.degree. C.
TABLE-US-00003 TABLE 3 SARS Nucleocapsid (NC) Peptides SEQ ID Cat #
NO: Peptide Sequence 9539 1 MSDNGPQSNQRSAPRI 9540 2
QSNQRSAPRITFGGPTDS 9541 3 RITFGGPTDSTDNNQNGGR 9542 4
STDNNQNGGRNGARPKQR 9543 5 GRNGARPKQRRPQGL 9544 6 RPKQRRPQGLPNNTASWF
9545 7 GLPNNTASWFTALTQHGK 9546 8 WFTALTQHGKEELRFPR 9547 9
HGKEELRFPRGQGVPI 9548 10 RFPRGQGVPINTNSGPDDQI 9549 11
NTNSGPDDQIGYYRRATR 9550 12 QIGYYRRATRRVRGGDGK 9551 13
TRRVRGGDGKMKELSPRW 9552 14 GKMKELSPRWYFYYL 9553 15
LSPRWYFYYLGTGPEASL 9554 16 YLGTGPEASLPYGANK 9555 17
EASLPYGANKEGIVWVA 9556 18 ANKEGIVWVATEGAL 9557 19 IVWVATEGALNTPKDHI
9558 20 GALNTPKDHIGTRNPNNNA 9559 21 IGTRNPNNNAATVLQL 9560 22
NNNAATVLQLPQGTTLPK 9561 23 QLPQGTTLPKGFYAEGSR 9562 24
PKGFYAEGSRGGSQASSR 9563 25 SRGGSQASSRSSSRSR 9564 26
ASSRSSSRSRGNSRNST 9565 27 RSRGNSRNSTPGSSR 9566 28
SRNSTPGSSRGNSPARMA 9567 29 SRGNSPARMASGGGETAL 9569 30
ALALLLLDRLNQLESKV 9570 31 DRLNQLESKVSGKGQQQQ 9571 32
KVSGKGQQQQGQTVTKK 9572 33 QQQGQTVTKKSAAEASKK 9573 34
KKSAAEASKKPRQKRTA 9574 35 SKKPRQKRTATKQYNV 9575 36
KRTATKQYNVTQAFGRR 9576 37 YNVTQAFGRRGPEQTQGNF 9577 38
RGPEQTQGNFGDQDLIR 9578 39 GNFGDQDLIRQGTDYKHW 9579 40
IRQGTDYKHWPQIAQFA 9580 41 KHWPQIAQFAPSASAFF 9581 42
QFAPSASAFFGMSRIGM 9582 43 AFFGMSRIGMEVTPSGTW 9583 44
GMEVTPSGTWLTYHGAIK 9584 45 TWLTYHGAIKLDDKDPQF 9585 46
IKLDDKDPQFKDNVILL 9586 47 PQFKDNVILLNKHIDAYK 9587 48
LLNKHIDAYKTFPPTEPK 9588 49 YKTFPPTEPKKDKKKK 9589 50
TEPKKDKKKKTDEAQPL 9590 51 KKKTDEAQPLPQRQKK 9591 52
AQPLPQRQKKQPTVTLL 9592 53 QKKQPTVTLLPAADMDDF 9594 54
LLPAADMDDFSRQLQNSM 9595 55 DFSRQLQNSMSGASA
TABLE-US-00004 TABLE 4 SARS Spike (5) Peptides SEQ ID Cat # NO:
Peptide Sequence 9597 56 MFIFLLFLTLTSGSDLDR 9598 57
TLTSGSDLDRCTTFDDV 9599 58 LDRCTTFDDVQAPNYTQH 9601 59
QHTSSMRGVYYPDEIFR 9602 60 GVYYPDEIFRSDTLYL 9603 61
EIFRSDTLYLTQDLFLPF 9604 62 YLTQDLFLPFYSNVTGFH 9605 63
PFYSNVTGFHTINHTF 9606 64 TGFHTINHTFGNPVIPFK 9607 65
TFGNPVIPFKDGIYFAA 9608 66 PFKDGIYFAATEKSNVVR 9609 67
AATEKSNVVRGWVFGSTM 9610 68 VRGWVFGSTMNNKSQSVI 9611 69
TMNNKSQSVIIINNSTNV 9612 70 VIIINNSTNVVIRACNF 9614 71
NFELCDNPFFAVSKPM 9615 72 NPFFAVSKPMGTQTHTMI 9616 73 PMGTQTHTMIFDNAF
9619 74 YISDAFSLDVSEKSGNFK 9620 75 DVSEKSGNFKHLREFVFK 9621 76
FKHLREFVFKNKDGFLYV 9622 77 FKNKDGFLYVYKGYQPI 9623 78
LYVYKGYQPIDVVRDL 9624 79 YQPIDVVRDLPSGFNTLK 9625 80
DLPSGFNTLKPIFKLPL 9626 81 TLKPIFKLPLGINITNFR 9627 82
PLGINITNFRAILTAF 9628 83 TNFRAILTAFSPAQDIW 9629 84
TAFSPAQDIWGTSAAAYF 9631 85 AAAYFVGYLKPTTFMLKY 9632 86
LKPTTFMLKYDENGTI 9633 87 MLKYDENGTITDAVDCSQ 9634 88
TITDAVDCSQNPLAELK 9636 89 LKCSVKSFEIDKG1Y 9637 90
KSFEIDKGIYQTSNFRVV 9638 91 IYQTSNFRVVPSGDVVRF 9639 92
VVPSGDVVRFPNITNL 9640 93 VVRFPNITNLCPFGEVF 9641 94
TNLCPFGEVFNATKFPSV 9642 95 VFNATKFPSVYAWERKKI 9643 96
SVYAWERKKISNCVADY 9644 97 KKISNCVADYSVLYNSTF 9645 98
DYSVLYNSTFFSTFKCY 9646 99 STFFSTFKCYGVSATKL 9647 100
KCYGVSATKLNDLCFSNV 9648 101 KLNDLCFSNVYADSFVVK 9649 102
NVYADSFVVKGDDVRQIA 9650 103 VKGDDVRQIAPGQTGVIA 9651 104
IAPGQTGVIADYNYKL 9652 105 GVIADYNYKLPDDFMGCV 9653 106
KLPDDFMGCVLAWNTRNI 9655 107 NIDATSTGNYNYKYRYLR 9656 108
NYNYKYRYLRHGKLRPF 9657 109 YLRHGKLRPFERDISNV 9658 110
RPFERDISNVPFSPDGK 9659 111 SNVPFSPDGKPCTPPAL 9660 112
DGKPCTPPALNCYWPL 9661 113 PPALNCYWPLNDYGFY 9663 114
GFYTTTGIGYQPYRVVVL 9665 115 VVLSFELLNAPATVCGPK 9666 116
NAPATVCGPKLSTDLIK 9667 117 GPKLSTDLIKNQCVNFNF 9668 118
IKNQCVNFNFNGLTGTGV 9669 119 NFNGLTGTGVLTPSSKRF 9670 120
GVLTPSSKRFQPFQQFGR 9671 121 RFQPFQQFGRDVSDF 9672 122
QQFGRDVSDFTDSVRDPK 9673 123 DFTDSVRDPKTSEILDI 9674 124
DPKTSEILDISPCSFGGV 9675 125 DISPCSFGGVSVITPGTNA 9676 126
VSVITPGTNASSEVAVLY 9678 127 LYQDVNCTDVSTAIHA 9679 128
CTDVSTAIHADQLTPAWR 9680 129 HADQLTPAWRIYSTGNNV 9681 130
WRIYSTGNNVFQTQAGCL 9682 131 NVFQTQAGCLIGAEHV 9684 132
HVDTSYECDIPIGAGICA 9685 133 DIPIGAGICASYHTVSLL 9686 134
CASYHTVSLLRSTSQKSI 9687 135 LLRSTSQKSTVAYTMSL 9688 136
KSIVAYTMSLGADSSIAY 9689 137 SLGADSSIAYSNNTIAI 9690 138
IAYSNNTIAIPTNFSISI 9691 139 AIPTNFSISITTEVMPV 9692 140
ISITTEVMPVSMAKTSV 9694 141 KTSVDCNMYICGDSTECA 9697 142
LQYGSFCTQLNRALSGIA 9698 143 QLNRALSGIAAEQDRNTR 9699 144
IAAEQDRNTREVFAQVK 9700 145 NTREVFAQVKQMYKTPTL 9701 146
VKQMYKTPTLKYFGGFNF 9702 147 TLKYFGGFNFSQILPDPL 9703 148
NFSQILPDPLKPTKRSFI 9704 149 PLKPTKRSFIEDLLFNKV 9705 150
FIEDLLFNKVTLADAGFM 9706 151 KVTLADAGFMKQYGECL 9707 152
GFMKQYGECLGDTNARDL 9708 153 CLGDTNARDLICAQKF 9709 154
ARDLICAQKFNGLTVL 9710 155 AQKFNGLTVLPPLLTDDM 9711 156
VLPPLLTDDMIAAYTAAL 9713 157 AALVSGTATAGWTFGAGA 9715 158
GAALQIPFAMQMAYRF 9716 159 PFAMQMAYRFNGIGV 9718 160
GIGVTQNVLYENQKQIA 9719 161 VLYENQKQIANQFNKAI 9721 162
KAISQIQESLTTTSTAL 9723 163 TALGKLQDVVNQNAQAL 9725 164
QALNTLVKQLSSNFGAI 9727 165 AISSVLNDILSRLDKVEA 9728 166
ILSRLDKVEAEVQIDRLI 9731 167 SLQTYVTQQLIRAAEIRA 9732 168
QLIRAAEIRASANLAATK 9733 169 RASANLAATKMSECVL 9734 170
AATKMSECVLGQSKRVDF 9735 171 VLGQSKRVDFCGKGYHLM 9736 172
DFCGKGYHLMSFPQAAPH 9737 173 LMSFPQAAPHGVVFLHV 9738 174
APHGVVFLHVTYVPSQER 9739 175 HVTYVPSQERNFTTAPAI 9740 176
ERNFTTAPAICHEGKAYF 9741 177 AICHEGKAYFPREGVFVF
9742 178 YFPREGVFVFNGTSWFI 9743 179 FVFNGTSWFITQRNFF 9744 180
SWFITQRNFFSPQII 9745 181 QRNFFSPQIITTDNTFV 9748 182
VIGIINNTVYDPLQPEL 9749 183 TVYDPLQPELDSFKEEL 9750 184
PELDSFKEELDKYFKNH 9751 185 EELDKYFKNHTSPDVDL 9752 186
KNHTSPDVDLGDISGINA 9753 187 DLGDISGTNASVVNIQK 9754 188
INASVVNIQKEIDRLNEV 9755 189 QKEIDRLNEVAKNLNESL 9756 190
EVAKNLNESLIDLQELGK 9757 191 SLIDLQELGKYEQYIKW 9758 192
LGKYEQYIKWPWYVWLGF 9759 193 KWPWYVWLGFIAGLIAIV 9760 194
GFIAGLIAIVMVTILL 9761 195 IAIVMVTILLCCMTSCCSCL 9762 196
CCMTSCCSCLKGACSCGS 9763 197 CLKGACSCGSCCKFDEDD 9764 198
GSCCKFDEDDSEPVLKGV 9765 199 DDSEPVLKGVKLHYT
[0207] 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
199116PRTArtificial SequenceNote Artificial = synthetic construct
1Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile1 5
10 15218PRTArtificial SequenceNote Artificial = synthetic construct
2Gln Ser Asn Gln Arg Ser Ala Pro Arg Ile Thr Phe Gly Gly Pro Thr1 5
10 15Asp Ser319PRTArtificial SequenceNote Artificial = synthetic
construct 3Arg Ile Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn
Gln Asn1 5 10 15Gly Gly Arg418PRTArtificial SequenceNote Artificial
= synthetic construct 4Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn
Gly Ala Arg Pro Lys1 5 10 15Gln Arg515PRTArtificial SequenceNote
Artificial = synthetic construct 5Gly Arg Asn Gly Ala Arg Pro Lys
Gln Arg Arg Pro Gln Gly Leu1 5 10 15618PRTArtificial SequenceNote
Artificial = synthetic construct 6Arg Pro Lys Gln Arg Arg Pro Gln
Gly Leu Pro Asn Asn Thr Ala Ser1 5 10 15Trp Phe718PRTArtificial
SequenceNote Artificial = synthetic construct 7Gly Leu Pro Asn Asn
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His1 5 10 15Gly
Lys817PRTArtificial SequenceNote Artificial = synthetic construct
8Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Glu Leu Arg Phe Pro1 5
10 15Arg916PRTArtificial SequenceNote Artificial = synthetic
construct 9His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln Gly Val
Pro Ile1 5 10 151020PRTArtificial SequenceNote Artificial =
synthetic construct 10Arg Phe Pro Arg Gly Gln Gly Val Pro Ile Asn
Thr Asn Ser Gly Pro1 5 10 15Asp Asp Gln Ile201118PRTArtificial
SequenceNote Artificial = synthetic construct 11Asn Thr Asn Ser Gly
Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala1 5 10 15Thr
Arg1218PRTArtificial SequenceNote Artificial = synthetic construct
12Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Val Arg Gly Gly Asp1
5 10 15Gly Lys1318PRTArtificial SequenceNote Artificial = synthetic
construct 13Thr Arg Arg Val Arg Gly Gly Asp Gly Lys Met Lys Glu Leu
Ser Pro1 5 10 15Arg Trp1415PRTArtificial SequenceNote Artificial =
synthetic construct 14Gly Lys Met Lys Glu Leu Ser Pro Arg Trp Tyr
Phe Tyr Tyr Leu1 5 10 151518PRTArtificial SequenceNote Artificial =
synthetic construct 15Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly
Thr Gly Pro Glu Ala1 5 10 15Ser Leu1616PRTArtificial SequenceNote
Artificial = synthetic construct 16Tyr Leu Gly Thr Gly Pro Glu Ala
Ser Leu Pro Tyr Gly Ala Asn Lys1 5 10 151717PRTArtificial
SequenceNote Artificial = synthetic construct 17Glu Ala Ser Leu Pro
Tyr Gly Ala Asn Lys Glu Gly Ile Val Trp Val1 5 10
15Ala1815PRTArtificial SequenceNote Artificial = synthetic
construct 18Ala Asn Lys Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala
Leu1 5 10 151917PRTArtificial SequenceNote Artificial = synthetic
construct 19Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys
Asp His1 5 10 15Ile2019PRTArtificial SequenceNote Artificial =
synthetic construct 20Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly
Thr Arg Asn Pro Asn1 5 10 15Asn Asn Ala2116PRTArtificial
SequenceNote Artificial = synthetic construct 21Ile Gly Thr Arg Asn
Pro Asn Asn Asn Ala Ala Thr Val Leu Gln Leu1 5 10
152218PRTArtificial SequenceNote Artificial = synthetic construct
22Asn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr Thr Leu1
5 10 15Pro Lys2318PRTArtificial SequenceNote Artificial = synthetic
construct 23Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala
Glu Gly1 5 10 15Ser Arg2418PRTArtificial SequenceNote Artificial =
synthetic construct 24Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly
Gly Ser Gln Ala Ser1 5 10 15Ser Arg2516PRTArtificial SequenceNote
Artificial = synthetic construct 25Ser Arg Gly Gly Ser Gln Ala Ser
Ser Arg Ser Ser Ser Arg Ser Arg1 5 10 152617PRTArtificial
SequenceNote Artificial = synthetic construct 26Ala Ser Ser Arg Ser
Ser Ser Arg Ser Arg Gly Asn Ser Arg Asn Ser1 5 10
15Thr2715PRTArtificial SequenceNote Artificial = synthetic
construct 27Arg Ser Arg Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser
Arg1 5 10 152818PRTArtificial SequenceNote Artificial = synthetic
construct 28Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro
Ala Arg1 5 10 15Met Ala2918PRTArtificial SequenceNote Artificial =
synthetic construct 29Ser Arg Gly Asn Ser Pro Ala Arg Met Ala Ser
Gly Gly Gly Glu Thr1 5 10 15Ala Leu3017PRTArtificial SequenceNote
Artificial = synthetic construct 30Ala Leu Ala Leu Leu Leu Leu Asp
Arg Leu Asn Gln Leu Glu Ser Lys1 5 10 15Val3118PRTArtificial
SequenceNote Artificial = synthetic construct 31Asp Arg Leu Asn Gln
Leu Glu Ser Lys Val Ser Gly Lys Gly Gln Gln1 5 10 15Gln
Gln3217PRTArtificial SequenceNote Artificial = synthetic construct
32Lys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys1
5 10 15Lys3318PRTArtificial SequenceNote Artificial = synthetic
construct 33Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu
Ala Ser1 5 10 15Lys Lys3417PRTArtificial SequenceNote Artificial =
synthetic construct 34Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro
Arg Gln Lys Arg Thr1 5 10 15Ala3516PRTArtificial SequenceNote
Artificial = synthetic construct 35Ser Lys Lys Pro Arg Gln Lys Arg
Thr Ala Thr Lys Gln Tyr Asn Val1 5 10 153617PRTArtificial
SequenceNote Artificial = synthetic construct 36Lys Arg Thr Ala Thr
Lys Gln Tyr Asn Val Thr Gln Ala Phe Gly Arg1 5 10
15Arg3719PRTArtificial SequenceNote Artificial = synthetic
construct 37Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln
Thr Gln1 5 10 15Gly Asn Phe3817PRTArtificial SequenceNote
Artificial = synthetic construct 38Arg Gly Pro Glu Gln Thr Gln Gly
Asn Phe Gly Asp Gln Asp Leu Ile1 5 10 15Arg3918PRTArtificial
SequenceNote Artificial = synthetic construct 39Gly Asn Phe Gly Asp
Gln Asp Leu Ile Arg Gln Gly Thr Asp Tyr Lys1 5 10 15His
Trp4017PRTArtificial SequenceNote Artificial = synthetic construct
40Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe1
5 10 15Ala4117PRTArtificial SequenceNote Artificial = synthetic
construct 41Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser
Ala Phe1 5 10 15Phe4217PRTArtificial SequenceNote Artificial =
synthetic construct 42Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly
Met Ser Arg Ile Gly1 5 10 15Met4318PRTArtificial SequenceNote
Artificial = synthetic construct 43Ala Phe Phe Gly Met Ser Arg Ile
Gly Met Glu Val Thr Pro Ser Gly1 5 10 15Thr Trp4418PRTArtificial
SequenceNote Artificial = synthetic construct 44Gly Met Glu Val Thr
Pro Ser Gly Thr Trp Leu Thr Tyr His Gly Ala1 5 10 15Ile
Lys4518PRTArtificial SequenceNote Artificial = synthetic construct
45Thr Trp Leu Thr Tyr His Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro1
5 10 15Gln Phe4617PRTArtificial SequenceNote Artificial = synthetic
construct 46Ile Lys Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val
Ile Leu1 5 10 15Leu4718PRTArtificial SequenceNote Artificial =
synthetic construct 47Pro Gln Phe Lys Asp Asn Val Ile Leu Leu Asn
Lys His Ile Asp Ala1 5 10 15Tyr Lys4818PRTArtificial SequenceNote
Artificial = synthetic construct 48Leu Leu Asn Lys His Ile Asp Ala
Tyr Lys Thr Phe Pro Pro Thr Glu1 5 10 15Pro Lys4916PRTArtificial
SequenceNote Artificial = synthetic construct 49Tyr Lys Thr Phe Pro
Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys1 5 10
155017PRTArtificial SequenceNote Artificial = synthetic construct
50Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro1
5 10 15Leu5116PRTArtificial SequenceNote Artificial = synthetic
construct 51Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg Gln
Lys Lys1 5 10 155217PRTArtificial SequenceNote Artificial =
synthetic construct 52Ala Gln Pro Leu Pro Gln Arg Gln Lys Lys Gln
Pro Thr Val Thr Leu1 5 10 15Leu5318PRTArtificial SequenceNote
Artificial = synthetic construct 53Gln Lys Lys Gln Pro Thr Val Thr
Leu Leu Pro Ala Ala Asp Met Asp1 5 10 15Asp Phe5418PRTArtificial
SequenceNote Artificial = synthetic construct 54Leu Leu Pro Ala Ala
Asp Met Asp Asp Phe Ser Arg Gln Leu Gln Asn1 5 10 15Ser
Met5515PRTArtificial SequenceNote Artificial = synthetic construct
55Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser Ala1 5 10
155618PRTArtificial SequenceNote Artificial = synthetic construct
56Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu1
5 10 15Asp Arg5717PRTArtificial SequenceNote Artificial = synthetic
construct 57Thr Leu Thr Ser Gly Ser Asp Leu Asp Arg Cys Thr Thr Phe
Asp Asp1 5 10 15Val5818PRTArtificial SequenceNote Artificial =
synthetic construct 58Leu Asp Arg Cys Thr Thr Phe Asp Asp Val Gln
Ala Pro Asn Tyr Thr1 5 10 15Gln His5917PRTArtificial SequenceNote
Artificial = synthetic construct 59Gln His Thr Ser Ser Met Arg Gly
Val Tyr Tyr Pro Asp Glu Ile Phe1 5 10 15Arg6016PRTArtificial
SequenceNote Artificial = synthetic construct 60Gly Val Tyr Tyr Pro
Asp Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu1 5 10
156118PRTArtificial SequenceNote Artificial = synthetic construct
61Glu Ile Phe Arg Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu1
5 10 15Pro Phe6218PRTArtificial SequenceNote Artificial = synthetic
construct 62Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser Asn Val
Thr Gly1 5 10 15Phe His6316PRTArtificial SequenceNote Artificial =
synthetic construct 63Pro Phe Tyr Ser Asn Val Thr Gly Phe His Thr
Ile Asn His Thr Phe1 5 10 156418PRTArtificial SequenceNote
Artificial = synthetic construct 64Thr Gly Phe His Thr Ile Asn His
Thr Phe Gly Asn Pro Val Ile Pro1 5 10 15Phe Lys6517PRTArtificial
SequenceNote Artificial = synthetic construct 65Thr Phe Gly Asn Pro
Val Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala1 5 10
15Ala6618PRTArtificial SequenceNote Artificial = synthetic
construct 66Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser
Asn Val1 5 10 15Val Arg6718PRTArtificial SequenceNote Artificial =
synthetic construct 67Ala Ala Thr Glu Lys Ser Asn Val Val Arg Gly
Trp Val Phe Gly Ser1 5 10 15Thr Met6818PRTArtificial SequenceNote
Artificial = synthetic construct 68Val Arg Gly Trp Val Phe Gly Ser
Thr Met Asn Asn Lys Ser Gln Ser1 5 10 15Val Ile6918PRTArtificial
SequenceNote Artificial = synthetic construct 69Thr Met Asn Asn Lys
Ser Gln Ser Val Ile Ile Ile Asn Asn Ser Thr1 5 10 15Asn
Val7017PRTArtificial SequenceNote Artificial = synthetic construct
70Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys Asn1
5 10 15Phe7116PRTArtificial SequenceNote Artificial = synthetic
construct 71Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys
Pro Met1 5 10 157218PRTArtificial SequenceNote Artificial =
synthetic construct 72Asn Pro Phe Phe Ala Val Ser Lys Pro Met Gly
Thr Gln Thr His Thr1 5 10 15Met Ile7315PRTArtificial SequenceNote
Artificial = synthetic construct 73Pro Met Gly Thr Gln Thr His Thr
Met Ile Phe Asp Asn Ala Phe1 5 10 157418PRTArtificial SequenceNote
Artificial = synthetic construct 74Tyr Ile Ser Asp Ala Phe Ser Leu
Asp Val Ser Glu Lys Ser Gly Asn1 5 10 15Phe Lys7518PRTArtificial
SequenceNote Artificial = synthetic construct 75Asp Val Ser Glu Lys
Ser Gly Asn Phe Lys His Leu Arg Glu Phe Val1 5 10 15Phe
Lys7618PRTArtificial SequenceNote Artificial = synthetic construct
76Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly Phe Leu1
5 10 15Tyr Val7717PRTArtificial SequenceNote Artificial = synthetic
construct 77Phe Lys Asn Lys Asp Gly Phe Leu Tyr Val Tyr Lys Gly Tyr
Gln Pro1 5 10 15Ile7816PRTArtificial SequenceNote Artificial =
synthetic construct 78Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp
Val Val Arg Asp Leu1 5 10 157918PRTArtificial SequenceNote
Artificial = synthetic construct 79Tyr Gln Pro Ile Asp Val Val Arg
Asp Leu Pro Ser Gly Phe Asn Thr1 5 10 15Leu Lys8017PRTArtificial
SequenceNote Artificial = synthetic construct 80Asp Leu Pro Ser Gly
Phe Asn Thr Leu Lys Pro Ile Phe Lys Leu Pro1 5 10
15Leu8118PRTArtificial SequenceNote Artificial = synthetic
construct 81Thr Leu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile
Thr Asn1 5 10 15Phe Arg8216PRTArtificial SequenceNote Artificial =
synthetic construct 82Pro Leu Gly Ile Asn Ile Thr Asn Phe Arg Ala
Ile Leu Thr Ala Phe1 5 10 158317PRTArtificial SequenceNote
Artificial = synthetic construct 83Thr Asn Phe Arg Ala Ile Leu Thr
Ala Phe Ser Pro Ala Gln Asp Ile1 5 10 15Trp8418PRTArtificial
SequenceNote Artificial = synthetic construct 84Thr Ala Phe Ser Pro
Ala Gln Asp Ile Trp Gly Thr Ser Ala Ala Ala1 5 10 15Tyr
Phe8518PRTArtificial SequenceNote Artificial = synthetic construct
85Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met Leu1
5 10 15Lys Tyr8616PRTArtificial SequenceNote Artificial = synthetic
construct 86Leu Lys Pro Thr Thr Phe Met Leu Lys Tyr Asp Glu Asn Gly
Thr Ile1 5 10 158718PRTArtificial SequenceNote Artificial =
synthetic construct 87Met Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr
Asp Ala Val Asp Cys1 5 10 15Ser Gln8817PRTArtificial SequenceNote
Artificial = synthetic construct 88Thr Ile Thr Asp Ala Val Asp Cys
Ser Gln Asn Pro Leu Ala Glu Leu1 5 10 15Lys8915PRTArtificial
SequenceNote Artificial = synthetic construct 89Leu Lys Cys Ser Val
Lys Ser Phe Glu Ile Asp Lys Gly Ile Tyr1 5 10 159018PRTArtificial
SequenceNote Artificial = synthetic construct 90Lys Ser Phe Glu Ile
Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg1 5 10 15Val
Val9118PRTArtificial SequenceNote Artificial = synthetic construct
91Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val1
5 10 15Arg Phe9216PRTArtificial SequenceNote Artificial = synthetic
construct 92Val Val Pro Ser Gly Asp Val Val Arg Phe Pro Asn Ile Thr
Asn Leu1 5 10 159317PRTArtificial SequenceNote Artificial =
synthetic construct 93Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
Pro Phe Gly Glu Val1 5 10 15Phe9418PRTArtificial SequenceNote
Artificial = synthetic construct 94Thr Asn Leu Cys Pro Phe Gly Glu
Val Phe Asn Ala Thr Lys Phe Pro1
5 10 15Ser Val9518PRTArtificial SequenceNote Artificial = synthetic
construct 95Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu
Arg Lys1 5 10 15Lys Ile9617PRTArtificial SequenceNote Artificial =
synthetic construct 96Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser
Asn Cys Val Ala Asp1 5 10 15Tyr9718PRTArtificial SequenceNote
Artificial = synthetic construct 97Lys Lys Ile Ser Asn Cys Val Ala
Asp Tyr Ser Val Leu Tyr Asn Ser1 5 10 15Thr Phe9817PRTArtificial
SequenceNote Artificial = synthetic construct 98Asp Tyr Ser Val Leu
Tyr Asn Ser Thr Phe Phe Ser Thr Phe Lys Cys1 5 10
15Tyr9917PRTArtificial SequenceNote Artificial = synthetic
construct 99Ser Thr Phe Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Ala
Thr Lys1 5 10 15Leu10018PRTArtificial SequenceNote Artificial =
synthetic construct 100Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn
Asp Leu Cys Phe Ser1 5 10 15Asn Val10118PRTArtificial SequenceNote
Artificial = synthetic construct 101Lys Leu Asn Asp Leu Cys Phe Ser
Asn Val Tyr Ala Asp Ser Phe Val1 5 10 15Val Lys10218PRTArtificial
SequenceNote Artificial = synthetic construct 102Asn Val Tyr Ala
Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln1 5 10 15Ile
Ala10318PRTArtificial SequenceNote Artificial = synthetic construct
103Val Lys Gly Asp Asp Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Val1
5 10 15Ile Ala10416PRTArtificial SequenceNote Artificial =
synthetic construct 104Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp
Tyr Asn Tyr Lys Leu1 5 10 1510518PRTArtificial SequenceNote
Artificial = synthetic construct 105Gly Val Ile Ala Asp Tyr Asn Tyr
Lys Leu Pro Asp Asp Phe Met Gly1 5 10 15Cys Val10618PRTArtificial
SequenceNote Artificial = synthetic construct 106Lys Leu Pro Asp
Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg1 5 10 15Asn
Ile10718PRTArtificial SequenceNote Artificial = synthetic construct
107Asn Ile Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr1
5 10 15Leu Arg10817PRTArtificial SequenceNote Artificial =
synthetic construct 108Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg His
Gly Lys Leu Arg Pro1 5 10 15Phe10917PRTArtificial SequenceNote
Artificial = synthetic construct 109Tyr Leu Arg His Gly Lys Leu Arg
Pro Phe Glu Arg Asp Ile Ser Asn1 5 10 15Val11017PRTArtificial
SequenceNote Artificial = synthetic construct 110Arg Pro Phe Glu
Arg Asp Ile Ser Asn Val Pro Phe Ser Pro Asp Gly1 5 10
15Lys11117PRTArtificial SequenceNote Artificial = synthetic
construct 111Ser Asn Val Pro Phe Ser Pro Asp Gly Lys Pro Cys Thr
Pro Pro Ala1 5 10 15Leu11216PRTArtificial SequenceNote Artificial =
synthetic construct 112Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn
Cys Tyr Trp Pro Leu1 5 10 1511316PRTArtificial SequenceNote
Artificial = synthetic construct 113Pro Pro Ala Leu Asn Cys Tyr Trp
Pro Leu Asn Asp Tyr Gly Phe Tyr1 5 10 1511418PRTArtificial
SequenceNote Artificial = synthetic construct 114Gly Phe Tyr Thr
Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val1 5 10 15Val
Leu11518PRTArtificial SequenceNote Artificial = synthetic construct
115Val Val Leu Ser Phe Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly1
5 10 15Pro Lys11617PRTArtificial SequenceNote Artificial =
synthetic construct 116Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu
Ser Thr Asp Leu Ile1 5 10 15Lys11718PRTArtificial SequenceNote
Artificial = synthetic construct 117Gly Pro Lys Leu Ser Thr Asp Leu
Ile Lys Asn Gln Cys Val Asn Phe1 5 10 15Asn Phe11818PRTArtificial
SequenceNote Artificial = synthetic construct 118Ile Lys Asn Gln
Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr1 5 10 15Gly
Val11918PRTArtificial SequenceNote Artificial = synthetic construct
119Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Pro Ser Ser Lys1
5 10 15Arg Phe12018PRTArtificial SequenceNote Artificial =
synthetic construct 120Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln
Pro Phe Gln Gln Phe1 5 10 15Gly Arg12115PRTArtificial SequenceNote
Artificial = synthetic construct 121Arg Phe Gln Pro Phe Gln Gln Phe
Gly Arg Asp Val Ser Asp Phe1 5 10 1512218PRTArtificial SequenceNote
Artificial = synthetic construct 122Gln Gln Phe Gly Arg Asp Val Ser
Asp Phe Thr Asp Ser Val Arg Asp1 5 10 15Pro Lys12317PRTArtificial
SequenceNote Artificial = synthetic construct 123Asp Phe Thr Asp
Ser Val Arg Asp Pro Lys Thr Ser Glu Ile Leu Asp1 5 10
15Ile12418PRTArtificial SequenceNote Artificial = synthetic
construct 124Asp Pro Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys
Ser Phe Gly1 5 10 15Gly Val12519PRTArtificial SequenceNote
Artificial = synthetic construct 125Asp Ile Ser Pro Cys Ser Phe Gly
Gly Val Ser Val Ile Thr Pro Gly1 5 10 15Thr Asn
Ala12618PRTArtificial SequenceNote Artificial = synthetic construct
126Val Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val1
5 10 15Leu Tyr12716PRTArtificial SequenceNote Artificial =
synthetic construct 127Leu Tyr Gln Asp Val Asn Cys Thr Asp Val Ser
Thr Ala Ile His Ala1 5 10 1512818PRTArtificial SequenceNote
Artificial = synthetic construct 128Cys Thr Asp Val Ser Thr Ala Ile
His Ala Asp Gln Leu Thr Pro Ala1 5 10 15Trp Arg12918PRTArtificial
SequenceNote Artificial = synthetic construct 129His Ala Asp Gln
Leu Thr Pro Ala Trp Arg Ile Tyr Ser Thr Gly Asn1 5 10 15Asn
Val13018PRTArtificial SequenceNote Artificial = synthetic construct
130Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly1
5 10 15Cys Leu13116PRTArtificial SequenceNote Artificial =
synthetic construct 131Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile
Gly Ala Glu His Val1 5 10 1513218PRTArtificial SequenceNote
Artificial = synthetic construct 132His Val Asp Thr Ser Tyr Glu Cys
Asp Ile Pro Ile Gly Ala Gly Ile1 5 10 15Cys Ala13318PRTArtificial
SequenceNote Artificial = synthetic construct 133Asp Ile Pro Ile
Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser1 5 10 15Leu
Leu13418PRTArtificial SequenceNote Artificial = synthetic construct
134Cys Ala Ser Tyr His Thr Val Ser Leu Leu Arg Ser Thr Ser Gln Lys1
5 10 15Ser Ile13517PRTArtificial SequenceNote Artificial =
synthetic construct 135Leu Leu Arg Ser Thr Ser Gln Lys Ser Ile Val
Ala Tyr Thr Met Ser1 5 10 15Leu13618PRTArtificial SequenceNote
Artificial = synthetic construct 136Lys Ser Ile Val Ala Tyr Thr Met
Ser Leu Gly Ala Asp Ser Ser Ile1 5 10 15Ala Tyr13717PRTArtificial
SequenceNote Artificial = synthetic construct 137Ser Leu Gly Ala
Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala1 5 10
15Ile13818PRTArtificial SequenceNote Artificial = synthetic
construct 138Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr Asn
Phe Ser Ile1 5 10 15Ser Ile13917PRTArtificial SequenceNote
Artificial = synthetic construct 139Ala Ile Pro Thr Asn Phe Ser Ile
Ser Ile Thr Thr Glu Val Met Pro1 5 10 15Val14017PRTArtificial
SequenceNote Artificial = synthetic construct 140Ile Ser Ile Thr
Thr Glu Val Met Pro Val Ser Met Ala Lys Thr Ser1 5 10
15Val14118PRTArtificial SequenceNote Artificial = synthetic
construct 141Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp
Ser Thr Glu1 5 10 15Cys Ala14218PRTArtificial SequenceNote
Artificial = synthetic construct 142Leu Gln Tyr Gly Ser Phe Cys Thr
Gln Leu Asn Arg Ala Leu Ser Gly1 5 10 15Ile Ala14318PRTArtificial
SequenceNote Artificial = synthetic construct 143Gln Leu Asn Arg
Ala Leu Ser Gly Ile Ala Ala Glu Gln Asp Arg Asn1 5 10 15Thr
Arg14417PRTArtificial SequenceNote Artificial = synthetic construct
144Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val1
5 10 15Lys14518PRTArtificial SequenceNote Artificial = synthetic
construct 145Asn Thr Arg Glu Val Phe Ala Gln Val Lys Gln Met Tyr
Lys Thr Pro1 5 10 15Thr Leu14618PRTArtificial SequenceNote
Artificial = synthetic construct 146Val Lys Gln Met Tyr Lys Thr Pro
Thr Leu Lys Tyr Phe Gly Gly Phe1 5 10 15Asn Phe14718PRTArtificial
SequenceNote Artificial = synthetic construct 147Thr Leu Lys Tyr
Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp1 5 10 15Pro
Leu14818PRTArtificial SequenceNote Artificial = synthetic construct
148Asn Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser1
5 10 15Phe Ile14918PRTArtificial SequenceNote Artificial =
synthetic construct 149Pro Leu Lys Pro Thr Lys Arg Ser Phe Ile Glu
Asp Leu Leu Phe Asn1 5 10 15Lys Val15018PRTArtificial SequenceNote
Artificial = synthetic construct 150Phe Ile Glu Asp Leu Leu Phe Asn
Lys Val Thr Leu Ala Asp Ala Gly1 5 10 15Phe Met15117PRTArtificial
SequenceNote Artificial = synthetic construct 151Lys Val Thr Leu
Ala Asp Ala Gly Phe Met Lys Gln Tyr Gly Glu Cys1 5 10
15Leu15218PRTArtificial SequenceNote Artificial = synthetic
construct 152Gly Phe Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile
Asn Ala Arg1 5 10 15Asp Leu15316PRTArtificial SequenceNote
Artificial = synthetic construct 153Cys Leu Gly Asp Ile Asn Ala Arg
Asp Leu Ile Cys Ala Gln Lys Phe1 5 10 1515416PRTArtificial
SequenceNote Artificial = synthetic construct 154Ala Arg Asp Leu
Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu1 5 10
1515518PRTArtificial SequenceNote Artificial = synthetic construct
155Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp1
5 10 15Asp Met15618PRTArtificial SequenceNote Artificial =
synthetic construct 156Val Leu Pro Pro Leu Leu Thr Asp Asp Met Ile
Ala Ala Tyr Thr Ala1 5 10 15Ala Leu15718PRTArtificial SequenceNote
Artificial = synthetic construct 157Ala Ala Leu Val Ser Gly Thr Ala
Thr Ala Gly Trp Thr Phe Gly Ala1 5 10 15Gly Ala15816PRTArtificial
SequenceNote Artificial = synthetic construct 158Gly Ala Ala Leu
Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe1 5 10
1515915PRTArtificial SequenceNote Artificial = synthetic construct
159Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val1 5
10 1516017PRTArtificial SequenceNote Artificial = synthetic
construct 160Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln
Lys Gln Ile1 5 10 15Ala16117PRTArtificial SequenceNote Artificial =
synthetic construct 161Val Leu Tyr Glu Asn Gln Lys Gln Ile Ala Asn
Gln Phe Asn Lys Ala1 5 10 15Ile16217PRTArtificial SequenceNote
Artificial = synthetic construct 162Lys Ala Ile Ser Gln Ile Gln Glu
Ser Leu Thr Thr Thr Ser Thr Ala1 5 10 15Leu16317PRTArtificial
SequenceNote Artificial = synthetic construct 163Thr Ala Leu Gly
Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala1 5 10
15Leu16417PRTArtificial SequenceNote Artificial = synthetic
construct 164Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn
Phe Gly Ala1 5 10 15Ile16518PRTArtificial SequenceNote Artificial =
synthetic construct 165Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser
Arg Leu Asp Lys Val1 5 10 15Glu Ala16618PRTArtificial SequenceNote
Artificial = synthetic construct 166Ile Leu Ser Arg Leu Asp Lys Val
Glu Ala Glu Val Gln Ile Asp Arg1 5 10 15Leu Ile16718PRTArtificial
SequenceNote Artificial = synthetic construct 167Ser Leu Gln Thr
Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile1 5 10 15Arg
Ala16818PRTArtificial SequenceNote Artificial = synthetic construct
168Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala1
5 10 15Thr Lys16916PRTArtificial SequenceNote Artificial =
synthetic construct 169Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met
Ser Glu Cys Val Leu1 5 10 1517018PRTArtificial SequenceNote
Artificial = synthetic construct 170Ala Ala Thr Lys Met Ser Glu Cys
Val Leu Gly Gln Ser Lys Arg Val1 5 10 15Asp Phe17118PRTArtificial
SequenceNote Artificial = synthetic construct 171Val Leu Gly Gln
Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His1 5 10 15Leu
Met17218PRTArtificial SequenceNote Artificial = synthetic construct
172Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala1
5 10 15Pro His17317PRTArtificial SequenceNote Artificial =
synthetic construct 173Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly
Val Val Phe Leu His1 5 10 15Val17418PRTArtificial SequenceNote
Artificial = synthetic construct 174Ala Pro His Gly Val Val Phe Leu
His Val Thr Tyr Val Pro Ser Gln1 5 10 15Glu Arg17518PRTArtificial
SequenceNote Artificial = synthetic construct 175His Val Thr Tyr
Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro1 5 10 15Ala
Ile17618PRTArtificial SequenceNote Artificial = synthetic construct
176Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala1
5 10 15Tyr Phe17718PRTArtificial SequenceNote Artificial =
synthetic construct 177Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro
Arg Glu Gly Val Phe1 5 10 15Val Phe17817PRTArtificial SequenceNote
Artificial = synthetic construct 178Tyr Phe Pro Arg Glu Gly Val Phe
Val Phe Asn Gly Thr Ser Trp Phe1 5 10 15Ile17916PRTArtificial
SequenceNote Artificial = synthetic construct 179Phe Val Phe Asn
Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe1 5 10
1518015PRTArtificial SequenceNote Artificial = synthetic construct
180Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile1 5
10 1518117PRTArtificial SequenceNote Artificial = synthetic
construct 181Gln Arg Asn Phe Phe Ser Pro Gln Ile Ile Thr Thr Asp
Asn Thr Phe1 5 10 15Val18217PRTArtificial SequenceNote Artificial =
synthetic construct 182Val Ile Gly Ile Ile Asn Asn Thr Val Tyr Asp
Pro Leu Gln Pro Glu1 5 10 15Leu18317PRTArtificial SequenceNote
Artificial = synthetic construct 183Thr Val Tyr Asp Pro Leu Gln Pro
Glu Leu Asp Ser Phe Lys Glu Glu1 5 10 15Leu18417PRTArtificial
SequenceNote Artificial = synthetic construct 184Pro Glu Leu Asp
Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn1 5 10
15His18517PRTArtificial SequenceNote Artificial = synthetic
construct 185Glu Glu Leu Asp Lys Tyr
Phe Lys Asn His Thr Ser Pro Asp Val Asp1 5 10
15Leu18618PRTArtificial SequenceNote Artificial = synthetic
construct 186Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile
Ser Gly Ile1 5 10 15Asn Ala18717PRTArtificial SequenceNote
Artificial = synthetic construct 187Asp Leu Gly Asp Ile Ser Gly Ile
Asn Ala Ser Val Val Asn Ile Gln1 5 10 15Lys18818PRTArtificial
SequenceNote Artificial = synthetic construct 188Ile Asn Ala Ser
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn1 5 10 15Glu
Val18918PRTArtificial SequenceNote Artificial = synthetic construct
189Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu1
5 10 15Ser Leu19018PRTArtificial SequenceNote Artificial =
synthetic construct 190Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile
Asp Leu Gln Glu Leu1 5 10 15Gly Lys19117PRTArtificial SequenceNote
Artificial = synthetic construct 191Ser Leu Ile Asp Leu Gln Glu Leu
Gly Lys Tyr Glu Gln Tyr Ile Lys1 5 10 15Trp19218PRTArtificial
SequenceNote Artificial = synthetic construct 192Leu Gly Lys Tyr
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu1 5 10 15Gly
Phe19318PRTArtificial SequenceNote Artificial = synthetic construct
193Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala1
5 10 15Ile Val19416PRTArtificial SequenceNote Artificial =
synthetic construct 194Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
Val Thr Ile Leu Leu1 5 10 1519520PRTArtificial SequenceNote
Artificial = synthetic construct 195Ile Ala Ile Val Met Val Thr Ile
Leu Leu Cys Cys Met Thr Ser Cys1 5 10 15Cys Ser Cys
Leu2019618PRTArtificial SequenceNote Artificial = synthetic
construct 196Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala
Cys Ser Cys1 5 10 15Gly Ser19718PRTArtificial SequenceNote
Artificial = synthetic construct 197Cys Leu Lys Gly Ala Cys Ser Cys
Gly Ser Cys Cys Lys Phe Asp Glu1 5 10 15Asp Asp19818PRTArtificial
SequenceNote Artificial = synthetic construct 198Gly Ser Cys Cys
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys1 5 10 15Gly
Val19915PRTArtificial SequenceNote Artificial = synthetic construct
199Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr1 5
10 15
* * * * *