U.S. patent application number 09/935344 was filed with the patent office on 2003-05-15 for viral vaccine composition, process, and methods of use.
Invention is credited to Jira, Vic, Jirathitikal, Vichai.
Application Number | 20030092145 09/935344 |
Document ID | / |
Family ID | 26921510 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030092145 |
Kind Code |
A1 |
Jira, Vic ; et al. |
May 15, 2003 |
Viral vaccine composition, process, and methods of use
Abstract
A composition for treating or preventing virus-induced
infections is described, along with a process of producing the
composition and methods of the composition's use. The composition
comprises viral pathogen-infected cell or tissue, or malignantly or
immunologically aberrant cells or tissues which has been reduced
and/or denatured. The preferred composition is administered across
a mucosal surface of an animal suffering or about suffer from
infection. The composition is administered as preventive or
therapeutic vaccine.
Inventors: |
Jira, Vic; (El Monte,
CA) ; Jirathitikal, Vichai; (Chachoengsao,
TH) |
Correspondence
Address: |
BLANK ROME COMISKY & MCCAULEY LLP
THE FARRAGUT BUILDING
SUITE 1000
900 17TH STREET, NW
WASHINGTON
DC
20006
US
|
Family ID: |
26921510 |
Appl. No.: |
09/935344 |
Filed: |
August 23, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60227520 |
Aug 24, 2000 |
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Current U.S.
Class: |
435/173.3 ;
424/204.1; 424/206.1; 424/207.1; 424/208.1; 424/209.1; 424/211.1;
424/212.1; 424/214.1; 424/215.1; 424/216.1; 424/217.1; 424/218.1;
424/224.1; 424/225.1; 424/229.1; 424/232.1; 424/233.1; 424/234.1;
424/464; 435/236 |
Current CPC
Class: |
A61K 39/001 20130101;
A61K 39/015 20130101; A61K 39/0005 20130101; A61K 2039/541
20130101; A61K 2039/51 20130101; A61K 2039/5252 20130101; A61K
2039/542 20130101; C12N 2710/14034 20130101; C12N 2740/16034
20130101; C12N 2760/16134 20130101; A61K 39/04 20130101; A61K
2039/552 20130101; A61K 39/0006 20130101 |
Class at
Publication: |
435/173.3 ;
435/236; 424/464; 424/204.1; 424/206.1; 424/207.1; 424/234.1;
424/208.1; 424/209.1; 424/211.1; 424/212.1; 424/214.1; 424/215.1;
424/216.1; 424/217.1; 424/218.1; 424/224.1; 424/225.1; 424/229.1;
424/232.1; 424/233.1 |
International
Class: |
C12N 007/04; A61K
039/165; A61K 039/155; C12N 013/00; A61K 039/145; A61K 039/17; A61K
039/125; A61K 039/193; A61K 039/245; A61K 039/27; A61K 039/23; A61K
009/20 |
Claims
What is claimed is:
1. A multivalent antiviral vaccine comprising one or more
heat-inactivated viral antigens, wherein at least one viral antigen
is capable in producing an immune response in a host when said
vaccine is administered orally at a dose that is sufficient for
preventing or treating the viral disease in said host.
2. The viral antigen of claim 1 wherein said antigen is derived
from influenza virus, cytomegalovirus, avian leukosis-sarcoma
virus, Rous Sarcoma virus, Mammalian C-type Murine leukemia virus,
Feline leukemia virus, simian sarcoma virus, B-type Mouse mammary
tumor virus, D-type virus Mason-Pfizer monkey virus, simian AIDS
virus, Human T-cell leukemia virus, Simian T-cell leukemia virus,
bovine leukemia virus, Human immunodeficiency virus, Simian
immunodeficiency virus, Feline immunodeficiency virus, Visna/maedi
virus, Equine infectious anemia virus, Caprine
arthritis-encephalitis virus, spumavirus, foamy virus, endogenous
retrovirus, papilloma virus, respiratory syncytial virus,
poliomyelitis virus, pox virus, measles virus, arbor virus,
Coxsackie virus, herpes virus, hantavirus, hepatitis virus,
baculovirus, mumps virus, circovirus, arenavirus, rotavirus,
Colorado Tick Fever CTF virus, Eyach virus, Langat virus, Powassan
virus, Omsk hemorrhagic fever virus, Crimean-Congo hemorrhagic
fever virus, Yellow fever virus, Encephalitis virus, St. Louis
Encephalitis virus, Venezualan equine encephalitis virus, Western
equine encephalitis virus, Chikungunya virus, Japanese encephalitis
virus, West Nile virus, Kyasanur forest disease virus, Dengue fever
virus, California encephalitis virus, adenovirus, Korean
haemorrhagic fever virus, hantavirus, Argentine haemorrhagic fever
virus, Junin virus, Aujeszky disease virus, Pseudorabies virus,
Herpesvirus, Chikungunya virus, cowpox virus, ebolavirus, Ganjam
virus, herpesvirus simiae, Lassa fever virus, Louping ill virus,
Lymphocytic choriomeningitis virus, Marburg virus, Milkers nodule
virus, Newcastle disease virus, Omsk haemorrhagic fever virus, Orf
virus, Parvovirus, Poliovirus, Pseudorabies, Rabies virus, Rift
Valley fever virus, Russian Spring-Summer encephalitis virus, Sabia
virus, vaccinia virus, vesicular stomatitis virus, Western equine
encephalitis virus, or Yelllow fever virus.
3. A composition for the induction of immunity to a viral pathogen
in a host in need thereof, said composition comprising reduced
viral pathogen formulated as an oral pill.
4. The viral pathogen of claim 3 wherein said pathogen is selected
from a group consisting of influenza virus, cytomegalovirus, avian
leukosis-sarcoma virus, Rous Sarcoma virus, Mammalian C-type Murine
leukemia virus, Feline leukemia virus, simian sarcoma virus, B-type
Mouse mammary tumor virus, D-type virus Mason-Pfizer monkey virus,
simian AIDS virus, Human T-cell leukemia virus, Simian T-cell
leukemia virus, bovine leukemia virus, Human immunodeficiency
virus, Simian immunodeficiency virus, Feline immunodeficiency
virus, Visna/maedi virus, Equine infectious anemia virus, Caprine
arthritis-encephalitis virus, spumavirus, foamy virus, endogenous
retrovirus, papilloma virus, respiratory syncytial virus,
poliomyelitis virus, pox virus, measles virus, arbor virus,
Coxsackie virus, herpes virus, hantavirus, hepatitis virus,
baculovirus, mumps virus, circovirus, arenavirus, rotavirus,
Colorado Tick Fever CTF virus, Eyach virus, Langat virus, Powassan
virus, Omsk hemorrhagic fever virus, Crimean-Congo hemorrhagic
fever virus, Yellow fever virus, Encephalitis virus, St. Louis
Encephalitis virus, Venezualan equine encephalitis virus, Western
equine encephalitis virus, Chikungunya virus, Japanese encephalitis
virus, West Nile virus, Kyasanur forest disease virus, Dengue fever
virus, California encephalitis virus, adenovirus, Korean
haemorrhagic fever virus, hantavirus, Argentine haemorrhagic fever
virus, Junin virus, Aujeszky disease virus, Pseudorabies virus,
Herpesvirus, Chikungunya virus, cowpox virus, ebolavirus, Ganjam
virus, herpesvirus simiae, Lassa fever virus, Louping ill virus,
Lymphocytic choriomeningitis virus, Marburg virus, Milkers nodule
virus, Newcastle disease virus, Omsk haemorrhagic fever virus, Orf
virus, Parvovirus, Poliovirus, Pseudorabies, Rabies virus, Rift
Valley fever virus, Russian Spring-Summer encephalitis virus, Sabia
virus, vaccinia virus, vesicular stomatitis virus, Western equine
encephalitis virus, and Yelllow fever virus.
5. An immunogen formulated as an oral pill, wherein upon oral
administration, said immunogen retains the ability to elicit an
immune response in a host in need of immune response.
6. The immunogen of claim 5 is administered to the host without an
immune adjuvant.
7. The immunogen of claim 5 wherein said immunogen is selected from
a group consisting of immunogens derived from fungi influenza
virus, cytomegalovirus, avian leukosis-sarcoma virus, Rous Sarcoma
virus, Mammalian C-type Murine leukemia virus, Feline leukemia
virus, simian sarcoma virus, B-type Mouse mammary tumor virus,
D-type virus Mason-Pfizer monkey virus, simian AIDS virus, Human
T-cell leukemia virus, Simian T-cell leukemia virus, bovine
leukemia virus, Human immunodeficiency virus, Simian
immunodeficiency virus, Feline immunodeficiency virus, Visna/maedi
virus, Equine infectious anemia virus, Caprine
arthritis-encephalitis virus, spumavirus, foamy virus, endogenous
retrovirus, papilloma virus, respiratory syncytial virus,
poliomyelitis virus, pox virus, measles virus, arbor virus,
Coxsackie virus, herpes virus, hantavirus, hepatitis virus,
baculovirus, mumps virus, circovirus, arenavirus, rotavirus,
Colorado Tick Fever CTF virus, Eyach virus, Langat virus, Powassan
virus, Omsk hemorrhagic fever virus, Crimean-Congo hemorrhagic
fever virus, Yellow fever virus, Encephalitis virus, St. Louis
Encephalitis virus, Venezualan equine encephalitis virus, Western
equine encephalitis virus, Chikungunya virus, Japanese encephalitis
virus, West Nile virus, Kyasanur forest disease virus, Dengue fever
virus, California encephalitis virus, adenovirus, Korean
haemorrhagic fever virus, hantavirus, Argentine haemorrhagic fever
virus, Junin virus, Aujeszky disease virus, Pseudorabies virus,
Herpesvirus, Chikungunya virus, cowpox virus, ebolavirus, Ganjam
virus, herpesvirus simiae, Lassa fever virus, Louping ill virus,
Lymphocytic choriomeningitis virus, Marburg virus, Milkers nodule
virus, Newcastle disease virus, Omsk haemorrhagic fever virus, Orf
virus, Parvovirus, Poliovirus, Pseudorabies, Rabies virus, Rift
Valley fever virus, Russian Spring-Summer encephalitis virus, Sabia
virus, vaccinia virus, vesicular stomatitis virus, Western equine
encephalitis virus, and Yelllow fever virus.
8. The immune response of claim 5 wherein said immune response is
directed against viral infection.
9. The oral pill of claim 5 wherein the amount of immunogen
comprised in said pill is between about 0.0000001% to about 20% by
weight.
10. An oral composition suitable for treating or preventing a
pathogen-induced infection in a host, the composition comprising a
first component comprising a denatured antigen of an
infection-inducing pathogen and a second component comprising a
denatured tissue from a pathogen-infected host.
11. The oral composition of claim 10 said composition further
comprising calcium.
12. The oral composition of claim 11 said composition further
comprising magnesium.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present invention claims priority date from provisional
U.S. application No. 60/227,520 filed Aug. 24, 2000 the disclosure
of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the therapy and prophylaxis
of pathogen-induced infections. In particular the invention relates
to antiviral vaccines for oral administration.
BACKGROUND OF THE INVENTION
[0003] Infectious diseases are caused by a pathogenic
microorganism, like a virus, bacterium, fungus, or the like which
enters and propagates in a living body. Common strategies to treat
infectious diseases include the administration of an antimicrobial
drug (antiviral or antibiotic) to patients suffering from such
infections.
[0004] In many of cases the pathogenic microorganisms are
eradicated or subsided by the action of a chemotherapeutic agent.
However, in cases involving infections induced by human
immunodeficiency virus (HIV) or herpes virus, the causative agents
are not easily eradicated, if at all. Microorganisms may also
acquire resistance to drugs. Furthermore, chemotherapeutic agents
are generally toxic to varying degrees to patients. For example, an
anti-HIV drug, AZT, causes life-threatening toxicity and anemia in
about 70% of treated patients. Prolonged treatment results almost
invariably in emergence of drug-resistant HIV. As a result, known
antibiotics and antivirals have not been entirely satisfactory in
terms of their antimicrobial spectrum, antimicrobial activity,
behavior in the body, safety, or ability to induce the appearance
of drug-resistant microorganisms.
[0005] Alternatives to chemotherapy include immunotherapeutic
treatment, such as a vaccination. Administration of vaccines to
non-infected hosts is referred to as "preventive vaccinating" while
administration to already infected hosts has been referred to as
"therapeutic vaccination." The term "prevention" includes an
attempt to halt the occurrence of a disease or disorder before it
happens. The term "therapy" includes an attempt to alleviate the
disease or clinical condition of an afflicted subject. The term
"immunotherapeutic" includes an attempt to activate or modulate the
immune system of the host. The term "subject" or "host" as used
hereinafter generally means humans. However, other animal species
are equally regarded as suitable hosts or subjects such as for
example primates, horses, cows, pigs, sheep, goats, dogs, cats,
rodents, fowl, fish, shellfish, crustaceans, worms, etc.
[0006] Vaccines usually come in injectable forms and are thus
problematic for a widespread use, requiring specialized skills for
delivery. As of today very few oral vaccines are known. An example
of an oral vaccine is Sabin's attenuated polio vaccine which
contains live but less virulent virus. The effect of such oral
vaccines is less predictable due to the destructive nature of the
digestive milieu and potential danger of virus being reverted to
virulent form. It is recognized that on passage to the stomach, the
vaccine antigenic component(s) are rapidly inactivated by the
gastric pH and digestive enzymes, and thus systemic assimilation
through the gut wall is poor or non-existent.
[0007] Thus, there remains a long-felt need for better therapies or
a vaccine, which preserves its activity despite hostile environment
in the stomach and intestines. Such therapies are additionally
needs to be free of undesirable properties, such as patient
toxicity or even death, the inducement of drug resistance, and the
requirement of complicated routes or means of delivery.
[0008] The body of a living organism is composed of unit(s)
generally known as a "cell." Cells assemble in the body to form
what is known as a "tissue." Blood is considered a specialized form
of connective tissue being a part of the hematopoietic system.
Processed blood products, such as dried deer blood, have been
described as an alleged health-invigorating food supplement. See,
for example, U.S. Pat. No. 5,637,345 incorporated by reference
herein. Whatever its eventual use it is imperative that products be
obtained from blood of healthy donors and not from sick or infected
donors. Wary of causing or spreading infection,
pathogen-contaminated blood or tissue is generally discarded as
unsuitable for any use.
[0009] Modern vaccines are mostly "acellular" or "subunit" vaccines
in which the immunogen is composed of a single bacterial/viral
protein or fragment thereof. In some vaccines the immunogen is a
nucleic acid of the pathogen or modified or unmodified pathogen
itself.
[0010] The overwhelming majority of vaccines are administered by
injection because it is generally believed that oral administration
of a vaccine leads to its destruction in the digestive tract.
Nevertheless, the search of the U.S. PTO database for patents
containing in their claims keywords such as "vaccine" and "virus"
and "oral" results in about 40 patents. The review of such patents
reveals that almost every one of them teaches chemically or
physically modified forms of the antigen or immunogen, mainly aimed
to prevent their degradation in the stomach and gut. Commonly these
modifications include entrapment in indigestible polymeric
carriers, e.g., U.S. Pat. No. 6,096,291; co-administration of acid
neutralizing buffers, e.g., U.S. Pat. No. 5,932,223;
co-administration of oil emulsions U.S. Pat. No. 5,885,590;
co-administration of absorption enhancing compounds, e.g., U.S.
Pat. No. 5,653,987; orally-administered package that will only
dissolve in the host animal's gut, e.g., U.S. Pat. No 5,676,950;
and other complicated and time-consuming safeguarding efforts.
Other approaches include expression of foreign, animal antigen
(usually a single protein) in genetically modified transgenic
plants and oral administration of the recombinant antigen in form
of an edible plant, e.g., U.S. Pat. No. 5,889,189. However, in
these plants such antigens are not naturally occurring since by
definition the animal pathogen cannot infect vegetable tissues.
Similarly, U.S. Pat. No. 5,643,577 discloses an oral vaccine
comprising an influenza antigen associated with red blood cells.
Here again this virus is not natural to the host cell since
influenza virus is not capable of infecting red blood cells. Thus,
none of the known strategies teaches, discloses, or suggests a
composition comprising a pathogen or a plurality of antigens of a
pathogen and pathogen-infected tissue, e.g., infected blood, of an
animal.
[0011] The present inventor describes a simple vaccine comprising a
pathogenic antigen and pathogen-infected host tissue. The
composition of the invention overcomes the difficulties inherent in
prior delivery systems and serves to introduce health modifying
agents into and across the mucosal membrane of a human or animal
subject. The composition of the invention is administered through a
mucosal surface, e.g., enterally by an oral route, to provide
significant clinical benefit to infected subjects. In a preferred
embodiment, the vaccine is not subjected to any special
modification aimed at enhancing the antigen "survival" in a hostile
digestive milieu.
[0012] The innovative vaccine provides protection both by whole
body immunity as well as in the critically important mucosal
tissues, such as the cervix, uterus, and rectum where a pathogen
often first enters the body during sexual contact or other
transmucosal means into person to be infected. Because the
gastrointestinal mucosa is a vast interface between the body and
the environment, it is the main entry site for many antigens. In
tests on animals and patients, the vaccines induces strong
reactions by mucosal immune cells in the time frame required to
stop infection. At the same time, the vaccines also stimulate
strong humoral and cell-mediated reactions to fight pathogen in the
blood stream. Thus, as oral vaccines, they work right away on the
mucosal cells, then stimulate or down-modulate systemic immunity by
raising antibody responses and priming killer cells.
[0013] The present invention provides a surprisingly effective and
broadly applicable strategy for treating and preventing a variety
of microbial infections in diverse host organisms ranging from
humans to insects. The same composition is remarkably effective in
treating cancer and immune disorders of autoimmune and inflammatory
nature.
SUMMARY OF THE INVENTION
[0014] The subject of the invention is a pharmaceutical composition
which possess immunomodulatory and antiviral activity, as well as
the ability to reverse chemotherapeutic drug resistance.
[0015] More specifically, the invention contemplates a composition
suitable for treating an animal infected with a pathogen or
preventing a pathogen-induced infection, the composition comprising
a first component comprising denatured antigen of an
infection-inducing pathogen and a second component comprising
denatured tissue derived from a pathogen-infected animal.
[0016] The simplest operating form of the invention is the
composition comprises a denatured tissue derived from a
pathogen-infected animal and which contains pathogen and components
thereof. The donor pathogen-infected animal may be of the same or
different species as the animal to be treated or vaccinated.
Preferably, the antigen used in the composition comprises a cell, a
nucleic acid, a pathogen, an amino acid, an oligopeptide, a
peptide, a protein, a glycoprotein, a lipid, a lectin, an
oligosaccharide, a lipoprotein, or is an antigen derived therefrom.
While any tissue is useful for preparing the composition, a
preferred tissue is comprised of blood or components thereof. Blood
can be obtained from pathogen-infected subjects. It also can be
obtained from rejected blood batches that have been found
contaminated with a pathogen, e.g., HIV, (from Blood Banks or
organizations like the Red Cross) or from umbilical cord blood from
HIV-positive delivering mothers.
[0017] In one aspect the present invention provides a
non-infectious immunotherapeutic containing viral particles devoid
of outer envelope proteins or containing selected antigens isolated
from a virus. There is also provided a vaccine effective against
HIV. In other aspect, the immunogen is useful for immunizing an
individual in need thereof and who is infected by a virus including
HIV, so as to induce immunoprotective factors protective against
progression of the infection. In another aspect, the vaccine is
useful for vaccinating an individual not previously infected with
HIV in order to prevent subsequently acquired infection. In another
aspect, there is provided a method of rendering a viral immunogen
non-infectious. The immunogen may also be used to produce
antibodies for passive immunotherapy, alone or in conjunction with
active immunotherapy, in individuals infected with a retrovirus,
including HIV.
[0018] The invention also contemplates a composition for treating
or preventing a disease, especially a malignant disease in a host
afflicted or about to be afflicted with the same. In this situation
instead of pathogen the preferred antigen is derived from malignant
cells or tissues. Thus, present invention relates to prophylactic
and therapeutic methods of anti-tumor immunization. For example
these methods can cross-prime a mammalian host to natural MHC class
I or II restricted tumor antigens with tumor antigen. A primary
tumor is resected from the patient and a population of tumor cells
are cultured in vitro. These cultured tumor cells are optionally
loaded with an artificial target antigen. The tumor cells are then
inactivated and introduced into the patient. This priming can be
simultaneous or subsequent to a direct immunization of the patient
with the same or substantially the same artificial target antigen.
This method of coupled host immunization promotes a tumor specific
cell-mediated immune response against multiple, undefined natural
tumor antigens expressed on the unmodified tumor cell surface.
[0019] While the preferred vaccine is a multivalent, oral vaccine
more specifically-targeted vaccines consisting of one or few select
tumor antigens are also contemplated. Such tumor associated
antigens can comprise oncofetal antigens, melanoma MPG, melanoma
p97, carcinoma Neu oncogene product, members of the MAGE family,
the BAGE family, the DAGE/Prame family, the GAGE family, the RAGE
family, the SMAGE family, NAG, Tyrosinase,
N-acetylglucosaminyltransferase-V, CQA 72/4, Laminin-P1, Yale Col.
Sr. Factor, Urinary gonadotropin Peptide (UGP), hCG and chains
thereof, Melan-A/MART-1, PSA, carcinoembryonic antigen (CEA),
gp100, MUC-1, MUC-2, TRP-1, beta-catenin, MUM-1, CDK-4, TAG-72,
CA-125, AMFr, M-344, 19a21 1, erb-2, p15, p21 of ras, mutated p53,
Bcr/Abl breakpoint peptide, HER-2/neu, PD-41, TCSF, GA733-2, HPV16
E7 or E6, MZ2-E, B7.1, B7.2, HOM-MEL-40, HOM-MEL-55, NY-COL-2,
HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4, and
HOM-TES-11 or one or more genes or DNA sequences encoding same.
[0020] As an anti-tumor agent, the instant composition is useful in
treating solid tumors and malignancies of lymphoreticular origin.
For example, the composition's utility for treatment of solid
tumors includes: cancers of the head and neck, including squamous
cell carcinoma; lung cancer, including small cell and non-small
cell lung carcinoma; mediastinal tumors; esophageal cancer,
including squamous cell carcinoma and adenocarcinoma; pancreatic
cancer; cancer of the hepatobiliary system, including
hepatocellular carcinoma, cholangiocarcinoma, gall bladder
carcinoma and biliary tract carcinoma; small intestinal carcinoma,
including adenocarcinoma, sarcoma, lymphoma and carcinoids;
colorectal cancer, including collon carcinoma and rectal carcinoma;
metastatic carcinoma; cancers of the genitourinary system,
including ovarian cancer, uterine sarcoma, and renal cell,
ureteral, bladder, prostate, urethral, penile, testicular, vulvar,
vaginal, cervical, endometrial, and fallopian tube carcinoma;
breast cancer; endocrine system cancer; soft tissue sarcomas;
malignant mesotheliomas; skin cancer, including squamous cell
carcinoma, basal cell carcinoma and melanoma; cancer of the central
nervous system; malignant bone tumors; and plasma cell
neoplasms.
[0021] The route of administration may be any route. A preferred
mode of administration comprises an enteral route, including
mucosal administration of the composition. The method comprises
contacting a mucosal surface of the animal in need of such
composition with an effective amount of a denatured antigen derived
from the tissues of an animal infected with said pathogen. Suitable
mucosal surfaces include, but are not limited to, nasal, buccal,
oral, vaginal, cervical, ocular, auditory, pulmonary tract,
urethral, digestive tract, skin, mucocutaneous, anal, cloacal,
rectal surface, and the like.
[0022] The present invention includes any pathogen. Examples
include, but are not limited to, a virus, a viroid, a bacterium, a
rickettsia, an acoxiella, a prion, a mycoplasma, or a fungus.
Moreover, the virus can be influenza virus, cytomegalovirus, avian
leukosis-sarcoma virus (ALV), Rous Sarcoma virus (RSV), Mammalian
C-type Murine leukemia virus (MLV), Feline leukemia virus (FeLV),
simian sarcoma virus (SIS), B-type viruses like Mouse mammary tumor
virus (MMTV), D-type viruses like Mason-Pfizer monkey virus (MPMV),
Simian AIDS viruses (SRVs), HTLV-BLV group such as Human T-cell
leukemia virus (HTLV), Simian T-cell leukemia virus (STLV), bovine
leukemia virus (BLV). Lentivirinae comprise Human immunodeficiency
virus (HIV-1 and HIV-2), Simian immunodeficiency virus (SIV),
Feline immunodeficiency virus (FIV), Visna/maedi virus (VMV),
Equine infectious anemia virus (EIAV), Caprine
arthritis-encephalitis virus (CAEV). Spumavirinae or "Foamy
viruses" like Human (HSRV), Simian (SSRV), Feline (FSRV), Bovine
(BSRV), Murine (MSRV), endogenous retroviruses (ERV), papilloma
virus, respiratory syncytial virus, poliomyelitis virus, pox virus,
measles virus, arbor virus, Coxsackie virus, herpes virus,
hantavirus, hepatitis virus, Baculovirus, mumps virus, circovirus,
vichaivirus, arenavirus, or rotavirus. A bacteria may be a member
of the genus Neisseria, Aerobacter, Pseudomonas, Porphyromonas,
Salmonella, Escherichia, Pasteurella, Shigella, Bacillus,
Helibacter, Corynebacterium, Clostridium, Mycobacterium, Yersinia,
Staphylococcus; Bordetelia, Brucelia, Vibrio, Streptococcus,
Plasmodium, Schisostoma, Candida. Any microbial infections, which
are present and/or transmitted as Zoonoses, Cyclozoonoses,
Metazoonoses, Saprozoonoses, Anthropozoonoses, Zooanthropozoonoses
and Amphixenoses, are encompassed by the present invention. The
invention in addition to whole pathogens also encompasses a single
antigen or a plurality of antigens from such pathogens, e.g., HIV
antigens: gp160, gag, pol, Nef, Tat, and Rev; the malaria antigens:
CS protein and Sporozoite surface protein 2; the Hepatitis B
surface antigens: Pre-S1, Pre-S2, HBc Ag, and HBe Ag; the influenza
antigens: HA, NP and NA; Hepatitis A surface antigens; the Herpes
virus antigens: EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV
early protein product, cytomegalovirus gB, cytomegalovirus gH, and
IE protein gp72; the respiratory syncytial virus antigens: F
protein, G protein, and N protein or fragments thereof.
[0023] It is also an object of the present invention to provide
various processes of making the composition. A preferred process of
producing a pharmaceutical composition useful against pathogen
infection comprises reducing step, i.e., drying pathogen-infected
tissue and denaturing the resulting dried tissue substantially in
the form as it is. The denatured composition can be processed
further, if desired. The denaturation step can be conducted by
exposing the composition to heat, detergent, acid, or base. Other
exemplary steps in making for example an anti-virus composition
include but are not limited to culturing cells from a cell line on
a cell culture medium, inoculating the cells with the virus in the
presence of a viral multiplication medium, propagating and
multiplying the virus, harvesting cells and the viral
multiplication medium, optionally subjecting the composition to the
purification preparation like washing, ion-exchange chromatography,
adsorption chromatography and/or gel permeation, denaturing the
composition by physical and/or chemical means, and formulating and
incorporating the obtained composition in a pharmaceutical
form.
[0024] In accordance with the object of the invention a process is
contemplated which comprises rapid freezing and/or drying of
aqueous products, pharmaceutical products, biologicals, blood
products, tissues and the like. The drying step may include
freeze-drying, spray-drying, flash-drying, heat-drying, or
vacuum-drying.
[0025] It is a further object of the invention to provide uniform
freezing and freeze-drying of all containers or vials in a batch to
minimize product variation from vial to vial.
[0026] Where the composition is provided as a powder, its dosage
may varied according to the route of administration, the age, body
weight and condition of the patient, and the type of the disease.
Generally, the preferred daily dose of the composition itself
ranges from about 0.00001 to 150g per subject. Most preferably, a
daily dose of 0.02 to 5 g is given in 1 to 3 divided doses. Instead
of daily doses the composition can be given in less frequent
regimen, e.g., once every 3 days, once weekly, once monthly, once
every 3 months, or once every 6 months as needed. Doses given once
a year or less frequently as required by exigencies of the therapy
or patient's condition are also contemplated as equally
suitable.
[0027] For the above indications the dosage may also vary depending
on the mode of administration or on the type of therapy desired.
Generally, however, dosage levels between 0.0001 and 100 mg/kg body
weight daily are administered to patients to obtain effective
clinical benefit. Usually, dosage forms suitable for oral, nasal,
pulmonar or transdermal administration comprise from about 0.0001
mg to about 100 mg, preferably from about 0.001 mg to about 50 mg
of the composition admixed when necessary with a pharmaceutically
acceptable carrier or diluent. When administered transdermally the
composition can be delivered under conditions of both constant
current (iontophoresis) and constant voltage (electroporation).
[0028] Optionally, the pharmaceutical composition of the invention
may further comprise one or more compounds exhibiting a different
mechanism of activity, e.g., an antibiotic or antiviral or any
other pharmacologically active material as deemed necessary by the
practitioner.
[0029] For example, the composition can be used in combination with
any of current or future AIDS drugs. Examples of such drugs include
those belonging to reverse transcriptase, integrase, protease,
fusion, adhesion, chemokine and other inhibitors like herbal drugs,
interleukins like interferon or IL-2 and including but not limited
to ZIAGEN Abacavir; PREVEON Adefovir; AGENERASE Amprenavir;
RETROVIR Zidovudine; VIDEX Didanosine; HIVID Zalcitabine; ZERIT
Stavudine; SUSTIVA Efavirenz; EPIVIR Lamivudine; INVIRASE
Saquinavir; FORTOVASE CRIXIVAN Indinavir; NORVIR Ritonavir;
RESCRIPTOR Delavirdine; VIRACEPT Nelfinavir; and VIRAMUNE
Nevirapine as representative AIDS drugs.
[0030] The compositions of the invention can be administered to
humans and valuable animals or plants to prevent infection and
generally for restoring health.
[0031] These and other objects, advantages and purposes of the
invention will be appreciated from the following description and
accompanying drawings illustrating preferred embodiments of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 illustrates the prophylactic effect of the
composition on Baculovirus infection in cultivated shrimp.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Various microorganisms cause devastating diseases to the
host. This invention contemplates a simple preventive and/or
therapeutic measure against them. Those of particular concern are
following: Bacillus anthracis, Bordetella pertussis, Borrelia spp.,
Brucella spp., Campylobacter spp., Clostridium botulinum,
Clostridium difficile, Clostridium tetani, Corynebacterium
diphtheriae, Erysipelothrix rhusiopathiae, Escherichia coli,
Francisella tularensis, Haemophilus ducreyi, Helicobacter pylori,
Leptospira spp., Mycobacterium tuberculosis. M. bovis,
Mycobacterium leprae, Mycoplasma spp., Neisseria gonorrhoea,
Neisseria meningitidis, Pasteurella spp., Pseudomonas mallei
(Burkholderia mallei), Pseudomonas pseudomallei (Burkholderia
pseudomallei), Rochalimaea, Salmonella typhi, Salmonella spp. other
than S. typhi, Serratia marcescens, Shigella spp., Streptobacillus
moniliformis, Streptococcus spp., Treponema pallidum, Vibrio
cholerae, Yersinia pestis; Chlamydias, Coxiellas and Rickettsias:
Chlamydia psittaci, Chlamydia trachomatis, Coxiella burnetii,
Rickettsia mooseri, Rickettsia orientalis, Rickettsia prowazeki,
Rickettsia rickettsii; Viruses: Adenovirus, Korean haemorrhagic
fever virus, Hantavirus, Argentine haemorrhagic fever virus, Junin
virus, Aujeszky disease virus, Pseudorabies virus, Herpesvirus,
Chikungunya virus, Cowpox virus, Coxsackieviruses, Creutzfelt-Jacob
Disease agent (prion), Crimea Congo haemorrhagic fever virus,
Cytomegalovirus, Dengue virus, Eastern encephalitis virus,
Ebolavirus, Ganjam virus, Hantavirus, Hepatitis A virus, Hepatitis
B virus, Hepatitis C virus, Hepatitis G, Herpesvirus simiae (B
virus), Human immunodeficiency virus, Junin virus, Kyansanur Forest
disease virus, Lassa fever virus, Louping ill virus, Lymphocytic
choriomeningitis virus, Marburg virus, Milkers nodule virus,
Newcastle disease virus, Omsk haemorrhagic fever virus, Orf virus,
Parvovirus, Poliovirus, Pseudorabies, Rabies virus, Rift Valley
fever virus, Russian Spring-Summer encephalitis virus, Sabia virus,
St Louis encephalitis virus, Vaccinia virus, Venezuelan equine
encephalitis, Vesicular stomatitis virus, Western equine
encephalitis virus, Yelllow fever virus; Fungi: Aspergilllus spp.,
Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus
neoformans, Microsporum and Trichophyton spp., Histoplasma
capsulatum, Sporothrix schenkii; Endoparasites like Ancylostama,
Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris,
Toxascaris, Uncinaria, Trichuris, Dirofilaria, Toxocara, Necator,
Enterobius, Strongyloides and Wuchereria; Acanthamoeba and other
amoebae, Cryptosporidium, Fasciola, Hartmanella, Acanthamoeba,
Giardia lamblia, Isospora belli, Leishmania, Naegleria, Plasmodium
spp., Pneumocystis carinii, Schistosoma spp., Toxoplasma gondii,
and Trypanosoma spp., among many others.
[0034] To solve this important health issue, the present invention
provides a composition containing pathogenic antigen denatured
thermally and/or under acidic or alkaline conditions or other
chemical means and derived from infected tissues or cells of
animals of the same species as the animal intended to be treated.
In another embodiment the species of animals as the source of
tissue or cell are different.
[0035] One can readily contemplate a variety of infections and
clinical conditions that can be dealt with using the teachings of
the present invention. These infections include, but are not
limited to, Mediterranean fever, undulant fever, Malta fever,
contagious abortion, epizootic abortion, Bang's disease, Salmonella
food poisoning, enteric paratyphosis, Bacillary dysentery,
Pseudotuberculosis, plague, pestilential fever, Tuberculosis,
Vibrios, Circling disease, Weil's disease, Hemorrhagic jaundice
(Leptospira icterohaemorrhagiae), canicola fever (L. canicola),
dairy worker fever (L. hardjo), Relapsing fever, tick-borne
relapsing fever, spirochetal fever, vagabond fever, famine fever,
Lyme arthritis, Bannworth's syndrome, tick-borne
meningopolyneuritis, erythema chronicum migrans, Vibriosis,
Colibacteriosis, colitoxemia, white scours, gut edema of swine,
enteric paratyphosis, Staphylococcal alimentary toxicosis,
staphylococcal gastroenteritis, Canine Corona Virus (CCV) or canine
parvovirus enteritis, feline infectious peritonitis virus,
transmissible gastroenteritis (TGE) virus, Hagerman Redmouth
Disease (ERMD), Infectious Hematopoietic necrosis (IHN), porcine
Actinobacillus (Haemophilus) pleuropneumonia, Hansen's disease,
Streptotrichosis, Mycotic Dermatitis of Sheep, Pseudoglanders,
Whitmore's disease, Francis' disease, deer-fly fever, rabbit fever,
O'Hara disease, Streptobacillary fever, Haverhill fever, epidemic
arthritic erythema, sodoku, Shipping or transport fever,
hemorrhagic septicemia, Ornithosis, Parrot Fever, Chlamydiosis,
North American blastomycosis, Chicago disease, Gilchrist's disease,
Cat Scratch Fever, Benign Lymphoreticulosis, Benign nonbacterial
Lymphadenitis, Bacillary Angiomatosis, Bacillary Peliosis Hepatis,
Query fever, Balkan influenza, Balkan grippe, abattoir fever,
Tick-borne fever, pneumorickettsiosis, American Tick Typhus,
Tick-borne Typhus Fever, Vesicular Rickettsiosis, Kew Gardens
Spotted Fever, Flea-borne Typhus Fever, Endemic Typhus Fever, Urban
Typhus, Ringworm, Dermatophytosis, Tinea, Trichophytosis,
Microsporosis, Jock Itch, Athlete's Foot, Sporothrix schenckii,
dimorphic fungus, Cryptococcosis and histoplasmosis, Benign
Epidermal Monkeypox, BEMP, Herpesvirus simiae, Simian B Disease,
Type C lethargic encephalitis, Yellow fever, Black Vomit,
hantavirus pulmonary syndrome, Korean Hemorrhagic Fever,
Nephropathia Epidemica, Epidemic Hemorrhagic Fever, Hemorrhagic
Nephrosonephritis, lymphocytic choriomeningitis, California
encephalitis/La crosse encephalitis, African Hemorrhagic Fever,
Green or Vervet Monkey Disease, Hydrophobia, Lyssa, Infectious
hepatitis, Epidemic hepatitis, Epidemic jaundice, Rubeola,
Morbilli, Swine and Equine Influenza, Fowl Plague, Newcastle
disease, Piroplasmosis, toxoplasmosis, African Sleeping Sickness,
Gambian Trypanosomiasis, Rhodesian Trypanosomiasis, Chagas's
Disease, Chagas-Mazza Disease, South American Trypanosomiasis,
Entamoeba histolytica, Balantidial dysentery, cryptosporidiosis,
giardiasis, Cutaneous leishmaniasis: Chiclero ulcer, espundia,
pianbols, uta, and buba (in the Americas); oriental sore, Aleppo
boil (in the Old World); Bagdad boil, Delhi boil, Bauru ulcer,
Visceral leishmaniasis: kala-azar, Microsporidiosis, Anisakiasis,
Trichinosis, Angiostrongylosis, eosinophilic meningitis or
meningoencephalitis (A. cantonensis), abdominal angiostrongylosis
(A. costaricensis), Uncinariasis, Necatoriasis, Hookworm Disease,
Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis,
Strongyloidiasis, Trichostrongylosis, Ascaridiasis,
Diphyllobothriasis, Sparganosis, Hydatidosis, Hydatid Disease,
Echinococcus granulosis, Cystic hydatid disease, Tapeworm
Infection, Schistosoma and the like. Malignant diseases caused by
infectious pathogens are contemplated as well. The examples of such
diseases include for example Burkitt lymphoma caused by EBV, Rous
sarcoma caused by Rous retrovirus, Kaposi' sarcoma caused by herpes
virus type 8, adult T-cell leukemia caused by HTLV-I retrovirus, or
hairy cell leukemia caused by HTLV-II, and many other tumors and
leukemias caused by infectious agents and viruses.
[0036] In addition diseases which are not caused by a known
pathogen, especially malignant or immunological diseases are
contemplated as suitable for treating or preventing by the
composition of the invention. Nonlimiting examples of these
diseases comprise leukemias like acute leukemia, acute lymphocytic
leukemia, acute myelocytic leukemias like myeloblastic,
promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic
leukemia like chronic myelocytic or granulocytic leukemia, chronic
lymphocytic leukemia, polycythemia vera, Sezary cell leukemia,
lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple
myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,
solid tumors like sarcomas and carcinomas, fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
Kaposi's sarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, uterine cancer,
testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
melanoma, neuroblastoma, retinoblastoma, mycosis fungoides,
pagetoid reticulosis among many others.
[0037] Instant composition is equally suitable as an
immunomodulator for treating immune disorders like inflammatory and
autoimmune diseases. As used herein, the terms "immunomodulator"
and "immunomodulating" encompass the activity of enhancing or
restoring the subject's immune system, as evidenced by measurable
clinical parameters and/or the patient's improved ability to combat
infection or disease, and the ability to heal tissue. Hence,
immunomodulation encompasses improvement of the immune system due
to an immunodeficient state (for example, caused by removal of the
thymus), and/or an immunodepressed state (for example, caused by
exposure to radiation). Furthermore, the present invention provides
for modulation of the immune system by lowering blood parameters
and other indicia of the immune state if these indicia are
abnormally elevated or suppressed. The present invention
encompasses the therapeutic method of treating the immunodeficient,
immunodepressed or wrongly activated immune state per se, thus
providing prophylaxis against infection and disease, as well as a
treatment of infection, disease or wound indirectly by enhancing
the immune system. It is therefore an object of the present
invention to provide pharmaceutical compositions which have broad
immunomodulating activity, as well as activity for other uses such
as treatment of infections, disease and wounds (bums, frost bites,
and the like), enhancement of metabolic processes, and many other
uses.
[0038] Inflammation plays a critical role in elimination of foreign
substances. Generally, recognition components of the host's immune
system bind to epitopes of the foreign matter, activating an
amplification system that includes the complement cascade,
cytokines, the coagulation cascade, lipid mediators and amines
produced by mast cells. These activated systems and components
alter blood flow, increase vascular permeability, augment adherence
of circulating leukocytes to vascular endothelium, promote
migration of leukocytes into tissues, and stimulate leukocytes to
destroy the foreign substance. By providing orally foreign
substances incorporated in the present composition an inflammatory
reaction is substantially subsided. The suitable inflammatory
condition favorably treatable by present composition is an
immunoinflammatory condition like transplant rejection, sepsis,
ARDS, asthma, trauma, oxidative stress, nitric oxide-related
inflammatory reaction, cell death or apoptosis, Alzheimer's
disease, Parkinson's disease, neurodegenerative disease,
demyelinating disease, HIV dementia, tumor angiogenesis,
irradiation damage, drug allergy, ischemia, reperfusion,
periodontitis, gingivitis, rhinitis, allergic conjunctivitis,
eczema, anaphylaxis, restenosis, stroke, congestive heart failure,
endometriosis, atherosclerosis, endosclerosis, corneal ulceration,
retinopathy, wound healing, gout, acute phase response, meningitis,
migraine, malignant ascites, malignant pleural effusion,
scleroderma, cirrhosis, keloids, adhesions and hypertrophic scars,
ankylosing spondylitis, Burger's disease, periarteritis nodosum,
hemolytic anemia, aplastic anemia, pure red cell anemia, idiopathic
thrombocytopenia, urticaria, endometrial hyperplasia, benign
prostatic hypertrophy, proliferative vitreal retirropathy,
dysplasias, skin infection, pyoderma, furunculitis, cellulitis,
acne, infralymphatic infection, lymphangitis, gynecological
infection, pelvic inflammatory disease, cervicitis, vaginitis,
tubo-ovarian abscess, adnexal abscess, acute respiratory disease,
sinusitis, parsinusitus, periapical granuloma, bum, frost bite.
[0039] The development of immunologic responsiveness to self is
called autoimmunity. The development of autoimmunity usually
involves the breakdown or circumvention of self-tolerance and by
orally administering to a host the self-antigens will restore said
self-tolerance. It has been surprisingly discovered that upon oral
administration benign self-antigens involved in autoimmunity as
opposed to orally administered malignant or pathogenic antigens
will trigger beneficial immune tolerance reaction. Representative
autoimmune diseases include allergy, alopecia areata, ulcerative
colitis, Mooren's ulcer, psoriasis, systemic lupus erythematosus,
rheumatoid arthritis, bursitis, rheumatoid vasculitis, joint
rigidity, collagen disorders, periarteritis nodosum, Wegener's
granulomatosis, polyarteritis nodosa, chronic graft versus host
disease, Waldenstrom's macroglobulinemia, hyperviscosity syndrome,
monoclonal gammopathy of undetermined origin, POEMS syndrome,
myeloma, fibromyalgia, macroglobulinemia, cold agglutinin disease,
myasthenia gravis cryptogenic fibrosing alveolitis, reactive
arthritis, Reiter's arthritis, polymyositis, dermatomyositis,
localized scleroderma, cutaneous scleroderma, systemic scleroderma,
Sjorgen's syndrome, Raynaud's phenomenon, Behcet's disease,
Kawasaki's disease, Hashimoto's thyroiditis, Steven-Johnson
syndrome, idiopathic burning mouth, aphthous ulceration, idiopathic
sprue, hyperimmunoglobin E, sarcoidosis, antiglomerular renal
membrane disease, primary biliary sclerosis, primary sclerosing
cholangitis, ulcerative colitis, Crohn's disease, juvenile onset
diabetes, insulin dependent diabetes mellitis, juvenile
dermatomyositis, autoimmune or chronic hepatitis, multiple
sclerosis, idiopathic inflammatory bowel disease,
keratoconjunctivitis sicca, vernal keratoconjunctivarthritis,
sympathetic ophthalmia, uveitis, uveoretinites, glomerulonephritis,
unities, atopic dermatitis, epidermolysis bullosa, eczematous
dermatitises, seborrhoeic dermatitis, lichen planus, pemphigus,
bullous pemphigoid, cutaneous eosinophilia, acne, Darier's disease,
icthyosis.
[0040] The instant composition can be prepared by a variety of
different methods, which have commonly been utilized in food
industry to prepare blood meals or dried foods in general. The
various methods can comprise flash Dried (Ring Dried and Drum
Dried), Spray Dried, Freeze Dried, and Conventional Cooker Dried.
The term "drying" is a collective term that can describe several
different drying processes.
[0041] Typically, a large portion of the moisture (water) is
mechanically removed. The subsequent semi-solid mass is then
transferred to another compartment (flash dryer) where the more
tightly bound water is rapidly removed. One skilled in the art
would know how to properly adjust dryer to obtain high quality
product. These processes are extensively used in the rendering
industry. As with any ingredient, proper plant procedures and
optimum temperature control will positively influence the quality
of the blood meal produced. Properly processed Ring Dried Blood
Meal is the preferred method of drying by many nutritionists due to
its consistent quality. The Ring Dried process produces a product
that is almost black in color and should be of a very fine particle
size. Spray Drying is a process commonly used in the drying of whey
proteins and dried fats. Spray Dried Blood Meal produces a product
that is readily soluble in water. Therefore, it can be used in
products by patients who may have difficulty of swallowing solid
formulations of the composition. Spray Drying is an acceptable
processing method, but is less common and more expensive than Flash
Dried Blood Meal. Conventional Cooker Dried Blood Meal is the
oldest of the blood processing methods. This type of Blood Meal is
processed in a batch type rendering cooker and there may be a great
variation in the biological potency of the composition.
[0042] An alternative preparation process requires an acid
hydrolysis (e.g., HCl hydrolysis or carbon dioxide hydrolysis) to
produce a liquid product. The product is then condensed under
elevated temperatures and vacuum to remove excess moisture and
increase solids' concentration. The concentrated liquid is then
dried. The resulting product is a flowable dry powder, which has
significant levels of high quality soluble protein and fat.
Residual lipids in such preparation can be stabilized with an
antioxidant such as BHT, TBHQ, or Ethoxyquin to prevent the
formation of peroxides. In general, antioxidant addition will
ensure adequate storage life. Amongst antioxidants used, BHA and
TBHQ, at a concentration of about 30 ppm, are the most effective.
Oxidation of treated meats during a 5-week storage period at
4.degree. C. is delayed as monitored by the TBA test. Other
additives like butylated hydroxyanisole, BHA;
tertbutylhydroquinone, TBHQ; sodium tripolyphosphate, STPP; sodium
pyrophosphate, SPP; sodium hexametaphosphate, SHMP; disodium salt
of diethylenediaminetetraacetic acid, sodium EDTA; sodium
ascorbate, SA; and sodium hypophosphite, SHP; 2-thiobarbituric acid
(TBA), Ellagic Acid, Eugenol, Isoeugenol, Quercetin, Kaempferol,
Rutin, Cinnamic Acid, Coumaric Acid, Ferulic Acid, Caffeic Acid,
Vanillic Acid, Gallic Acid, Syringic Acid, Chlorogenic Acid, and
3,5dimethoxy-4-hydroxycinnamic acid (DMHC) among others.
[0043] By alternative feature of the novel composition of the
invention, the composition can be in form of emulsion-type product
and made into a food like frankfurters, wieners, or restructured
meats or slices or chunks of meat and pieces as well as large cuts,
prepared seafoods or other hybrid products and the cooked cured
meat or any of the variants. For example a process for producing a
food product simulating meat from an anticoagulant-treated,
coagulable blood fluid can be carried out by controlled,
chemically-stimulated coagulation of the blood fluid under
controlled temperature conditions. This can comprise coagulating an
anticoagulant-treated, coagulable blood fluid, whole animal blood,
a plasma phase of animal blood, a mixture of a hemoglobin phase of
animal blood and a plasma phase of animal blood, a mixture of whole
animal blood and a plasma phase of animal blood; a mixture of whole
animal blood and a hemoglobin phase of animal blood or a mixture of
whole animal blood, a plasma phase of animal blood and a hemoglobin
phase of animal blood, in the presence of an exudation-inhibiting
agent and a coagulation stimulant, both present in effective
amounts, while maintaining the blood fluid at a temperature within
about 10.degree. C., either above or below the physiological
temperature of the animal from which blood has been derived to
chemically induce coagulation, and then recovering the resultant
coagulate.
[0044] Where the vaccine is intended for animal or veterinary use,
the vaccine is conveniently administrable with the animal feed,
such as grain or food pellets, bait, or in the animal drinking
water. The vaccine composition may also be incorporated into a food
meal or may be topically applied, i.e., sprayed onto meal.
[0045] The medical preparation forms can be solid or fluid. In
addition, it is possible to administer the active agent with
vehicles, diluents and additives which are usual in
biopharmaceutics, are pharmacologically harmless and are compatible
with the active ingredient. Additives can include fillers,
dispersants, binding agents, moisteners, stabilizers, lubricants,
emulsifiers, sweeteners, flavors and similar. These additives can
additionally include for example melantine solutions, pectin
solutions, lactose, sodium chloride, talcum, starch, boric acid,
paraffin oil, paraffin, stearic acid and its derivatives, cocoa
butter, rubber, syrups, licorice extracts, yeast extracts, honey,
glycerol, silicious earth, kaolin, magnesium oxide, beeswax and
plant oils. Stabilizers, for example, can be added prior to
lyophilization these include but not limited to hydrolyzed gelatin,
sodium chloride, sodium bicarbonate, human serum albumin, cysteine,
sodium glutamate, chelator, sugars like sorbitol, mannitol,
dulcitol, sucrose, lactose, maltose or trehalose, and buffers like
phosphate or citrate. The use of such media and agents for
pharmaceutical active substances is well known in the art and is
described by way of example in Remington's Pharmaceutical Sciences,
18th Edition, Mack Publishing Company, Pennsylvania, U.S.A. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, use thereof in the vaccine compositions is
contemplated.
[0046] When the composition of the present invention can be used
directly for purposes of clinical therapy and prophylaxis it can
have the form of an oral preparation or a parenteral preparation.
The term "parenteral" includes subcutaneous, intravenous, epidural,
irrigation, intramuscular, release pumps, or infusion. Without
limiting the composition can be also administered
intra-articularly, intrasynovially, intrathecally, periostally,
intratumorally, peritumorally, intralesionally, perilesionally,
sublingually, buccally, transdermally, topically or by inhalation.
It also can be administered as a dressing for a wound or lesion.
However, oral administration of the composition is especially
preferred. For oral use, the composition of the present invention
can be used alone or in combination with pharmaceutically
acceptable carriers to form pharmaceutical preparations such as
capsules, pills, lozenges, tablets, dragees, sachets, tea bags,
granules, powders, coated tablets, sugar coated tablets, wafers,
sugar cubes, gels, hydrogels such as particles of a
hydrophilic-hygroscopic polysaccharide, foams, suppositories,
inhalants, juices, shakes, chewing gum, tooth paste, dentifrice,
mouthwash, candies, and emulsions.
[0047] Suitable pharmaceutical carriers include, for example,
fillers such as lactose, sucrose, mannitol, glucose, starch,
sorbitol, glycine, calcium phosphate and microcrystalline
cellulose; binders such as starch, casein, gelatin, acacia,
glucose, sucrose, sorbitol, mannitol, tragacanth,
hydroxypropylcellulose, hydroxypropoxymethylcellulose,
carboxymethylcellulose, 2-methyl-5-vinylpyridine/methyl
methacrylate/ethylacrylate copolymer, polyvinylpyrrolidone and
sodium alginate; alginate gel; lubricants such as stearic acid,
hardened oil, magnesium stearate, calcium stearate, polyoxyethylene
monostearate, talc, silicon oxide and polyethylene glycol;
disintegrators such as potato starch, and starch containing a
surfactant or the like; facilitators like magnesium sulphate; and
humectants such as sodium lauryl sulfate.
[0048] Composition of the present invention can also be
administered in the form of liposomes. As is known in the art,
liposomes or artificial lipid vesicles are generally derived from
phospholipids or other lipid substances. Additionally they can
contain muramyl peptide, a metabolizable oil, and optionally an
additional emulsifying agent. Liposomes are formed by mono- or
multi-lamellar hydrated liquid crystals that are dispersed in an
aqueous medium. A typical process for making a liposome preparation
comprised of liposomes that contain an encapsulated composition of
the invention, comprises hydrating a lipid or liposome formulation
with a solution of a material to be encapsulated; providing a
plurality of portions of a dry lipid or dry liposome formulation;
hydrating each of said plurality of portions with a solution
comprising said material to be encapsulated; and combining each of
said plurality of portions to form a single liposome preparation,
thereby forming a liposome preparation comprising liposomes that
contain said encapsulated material. Any non-toxic,
physiologically-acceptable and metabolizable lipid capable of
forming liposomes can be used. The present compositions in liposome
form can contain, in addition to a compound of the present
invention, stabilizers, preservatives, excipients, and the like.
The preferred lipids are the phospholipids and the phosphatidyl
cholines (lecithins), both natural and synthetic. Methods to form
liposomes are well known in the art. For example, cochleates
containing biologically relevant molecule component, a negatively
charged lipid component, and a divalent cation component. The
cochleate has an extended shelf life, even in a desiccated state.
Advantageously, the cochleate can be ingested.
[0049] Other encapsulating microspheres or vesicles are also known.
For example one embodiment is an autogenous vaccine composition
comprising protein-lipid vesicles, said protein-lipid vesicles
comprising a patient-specific antigen from a patient that is
infected with human immunodeficiency virus; immunomodulator which
is at least one member selected from the croup consisting of an
envelope protein of an animal or human virus, a chemical
immunomodulator, interferon alpha, interferon gamma, and
interleuken 12; and lipid that forms a protein-lipid vesicle;
wherein the patient-specific antigen is prepared by extraction from
infected cells, tissue or organs with non-ionic detergent in
physiologically balanced buffer.
[0050] Another type comprises a biodegradable-biocompatible
poly(DL-lactide-co-glycolides. The bulk matrix is produced by a
solvent evaporation process and antigen is pre-encapsulated into a
conformationally stabilizing hydrophilic matrix consisting of an
appropriate mono, di- or tri-saccharide or other carbohydrate
susbstance by lyophilization prior to its final encapsulation into
the microsphere by a solvent extraction process employing
acetonitrile as the polymer solvent, mineral oil as the emulsion's
external phase, and heptane as the extractant.
[0051] In yet another embodiment the polymer core matrix is formed
from at least two highly water soluble biodegradable polymers,
selected for example from starch, crosslinked starch, ficoll,
polysucrose, polyvinyl alcohol, gelatine, hydroxymethyl cellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-ethyl cellulose, hydroxypropyl-methyl cellulose,
sodium carboxymethyl cellulose, cellulose acetate, sodium alginate,
polymaleic anhydride esters, polyortho esters, polyethyteneimine,
polyethylene glycol, methoxypolyethylene glycol, ethoxypolyethylene
glycol, polyethylene oxide, poly(1,3 bis(p-carboxyphenoxy)
propane-co-sebacic anhydride, N,N-diethylaminoacetate, block
copolymers of polyoxyethylene and polyoxypropylene. The
microspheres are additionally coated with a (D,L lactide-glycolide)
copolymer.
[0052] The blood powder as one embodiment of the invention can be
conventionally put into gelatin hard capsules or formulated into
tablets having the instant composition at about 0.5mg or higher;
Magnesium stearate 5mg, optionally Corn starch at 20mg and Lactose
194.5mg. The tablet can contain other ingredients like vitamins,
antioxidants and alike. Although a preferred saccharide is lactose,
other saccharides can be used, such as sucrose, mannitol, glucose,
polyaspartic acid, inositol hexaphosphate (phytic acid), sialic
acid, N-acetylneuraminic acid-lactose, inositol, fructose, maltose
or galactose. In addition, combinations of saccharides can be used,
for example, lactose and mannitol, or sucrose and mannitol. Other
sugars like phosphorylated sugars are equally suitable. The
concentration of the saccharide can range from about 1% to 80% by
weight.
[0053] In the case of suppositories, either rectal or vaginal,
cacao butter, Witepsol, Subanal, polyethylene glycol, polypropylene
glycol, glycerogelatin, gelatin capsules and the like can be used
as bases. Other additives include well-known safe antiseptics such
as methyl p-hydroxybenzoate, paraben forms, boric acid, propyl
p-hydroxybenzoate, butyl p-hydroxybenzoate and butyl hydroxyanisol;
and safe colorants. The composition can also be administered as
enema or clysters or prepared in a such way that it will be orally
administered as a pharmaceutical preparation with colon selective
delivery.
[0054] In preparing an injection (solution, emulsion or
suspension), it is generally sterilized and is preferably made
isotonic to the blood. In preparing the solution, emulsion or
suspension, there can be used all diluents commonly used in the
art, such as water, ethyl alcohol, propylene glycol, ethoxylated
isostearyl alcohol, polyoxyisostearyl alcohol and polyoxyethylene
sorbitan-fatty acid esters. In this case, the injection may contain
sodium chloride, glucose or glycerine in an amount sufficient to
make the injection isotonic, and may further contain a solubilizing
agent, a buffer solution, a soothing agent, etc. all ordinarily
used. The injection may furthermore contain, as necessary, a
coloring agent, a preservative, a perfume, a flavoring agent, a
sweetening agent and other drugs.
[0055] A variety of buffers may be used, depending on the pH range
desired, preferably between 7.0 and 7.8. Suitable buffers include
phosphate buffer and citrate buffer.
[0056] The lyophilized or dehydrated composition of the subject
invention can be reconstituted to an aqueous solution using a
variety of substances, but are preferably reconstituted using
water. In certain instances, dilute salt solutions which bring the
final formulation to isotonicity can also be used. In other
circumstances solutions can be a fruit juice or milk. In addition,
it can be advantageous to use aqueous solutions containing
components known to enhance the activity of the reconstituted
composition. Such components can include cytokines, such as IL-2,
polycations, such as protamine sulfate, or other components, which
enhance the efficiency of the composition, e.g., vitamins and
minerals. Other enhancing compounds can be sodium salicylate,
sodium lauryl sulphate, disodium ethylenediaminetetraacetic acid
(disodium EDTA), oleic acid, linoleic acid, monoolein, lecithin,
lysolecithin, deoxycholate, sodium deoxycholate, chenodeoxycholate,
taurodeoxycholate, glycochenodeoxycholate, polyoxyethylene X-lauryl
ether wherein X is from 9 to 20, sodium tauro-24,
25-dihydrofusidate, polyoxyethylene ether, polyoxyethylene sorbitan
esters, p-t-octylphenoxypolyoxyethylene,
N-lauryl-.beta.-D-maltopyranoside, 1-dodecylazacycloheptane-2-azone
and phospholipids, wherein the amount of each of the enhancing
compounds is present in a concentration of from 1 to 10 w/w % of
the total formulation. Preferably each of the enhancing compounds
is present in a concentration of from 0.0005 to 3.5 w/w %
[0057] Lyophilized or dehydrated composition can be reconstituted
with any convenient volume of water or the reconstituting agents
noted above that allow substantial, and preferably total
solubilization of the lyophilized or dehydrated sample.
[0058] If the composition is an aqueous solution it can contain a
neutral salt which is used to adjust the final formulation to an
appropriate iso-osmotic salt concentration. Suitable neutral salts
include sodium chloride, potassium chloride or magnesium chloride.
A preferred salt is sodium chloride.
[0059] Since denatured antigen of this invention induces either
highly effective oral immune tolerance or stimulation, the
administration of the drugs and intake of the functional foods of
the invention can inhibit or stimulate immune responses and thus
useful for treating and preventing pathogen-caused infections.
[0060] For a convenience' sake, in the present specifications the
word "oral" includes intranasal, sublingual, bronchial, pulmonary,
enteral, parenteral, mucocutaneous, transdermal, and transmucosal
in addition to the original meaning of the word "oral".
[0061] The oral drugs and functional foods of the present invention
contain antigen or plurality of antigens denatured thermally or
chemically with the agents specifically and/or inherently
associated with a pathogen.
[0062] In the present invention, the antigen can be further
purified and isolated by conventional methods from raw materials.
For example, antigen is prepared by partial digestion of tissues
containing antigen with such proteases as pepsin and pronase, and
then purification by the differential salting-out method
(dialysis). Antigen that is commercially available is also useful.
Origins of antigen or tissue containing the antigen are not
necessarily restricted. Examples of the origins are as follows;
mammals as humans, primates, cattle, pigs, goats, sheep, horses,
rabbits, mice, and rats, birds as chicken, turkeys, and ostriches,
reptiles as turtles and snakes, and water-living animals like fish,
e.g., tuna, bonito, salmon, shark, trout, and ray, shell fish and
mollusks; whales and dolphins; insects like shrimp, bees, silk
worms, ticks, mosquitoes, and crickets. For example specific avian
pathogens are derived from poultry pathogens selected from the
group consisting of Newcastle disease virus (NDV), Infectious
bronchitis virus (IBV), Infectious bursal disease virus (IBDV),
Turkey rhinotracheitis virus (TRTV), Infectious laryngotracheitis
virus (ILTV), Egg drop syndrome (EDS) virus, avian
encephalomyelitis virus, reticuloendotheleisis virus, avian pox
viruses, avian adenoviruses, infectious coryza, fowl typhoid, fowl
cholera, Mycoplasma gallisepticum. E. coli and Salmonella.
[0063] Plant infecting pathogens are contemplated as well.
Exemplary plant viruses are potexviruses (e.g., PVX), carlaviruses
(e.g., PVM), tobraviruses (e.g., TRV, PEBV, PRV), tobamoviruses
(e.g., TMV, ToMV, PPMV), luteoviruses (e.g., PLRV), potyviruses
(e.g., TEV, PPV, PVY), tombusviruses (e.g., CyRSV), nepoviruses
(e.g., GFLV), bromoviruses (e.g., BMV), and topamoviruses. These
viruses along with infected tissues are made into fertilizers and
are then fed to plants in such manner that allows their absorption
into target plant. The compound may also be used against nematodes
occurring in the soil or parasitic to plants.
[0064] The antigen can be synthesized chemically based on the
information about the amino acid or nucleic acid sequences of the
antigen of the above-described animal species or by recombinant DNA
technology.
[0065] For example, an AIDS vaccine can be made comprising a vector
which would contain a gag gene, env gene or pol gene of HIV
retrovirus under the control of a promoter. The steps in making
vector can comprise a step in which a gene fusion is introduced
into a host cell, said gene fusion comprising a hybrid DNA molecule
which is produced by inserting or fusing the DNA encoding an
immunogen into DNA encoding said support and which is fused with a
promoter. DNA vector can be derived from a plasmid, a
bacteriophage, a virus and/or a cosmid. Viral genes can be
assembled together or placed into a vector individually. An
exemplary vector can be a live pseudorabies virus, herpes virus, or
adenovirus which is able to propagate in a non-complementing cell
culture and which produces non-infectious progeny virions, wherein
the vector harbors a gene encoding an antigen of a virus not
necessarily associated with antibody-dependent enhancement (ADE) of
viral infectivity. Other non-infectious, retrovirus-like particles
can comprise an assembly of an env gene product, a pol gene product
and a gag gene product contain an antigenic marker which is
non-retroviral or non-HIV retroviral. In one embodiment, the marker
comprises an amino acid sequence containing an epitope inserted
into the gag gene product at an antigenically-active insertion
site. In another embodiment, the marker comprises an antigenic
anchor sequence operatively connected to the env gene product
replacing endogenous anchoring function. The non-infectious,
retrovirus-like particles have utility in in vivo administration
including to humans and in diagnosis. The presence of the antigenic
marker enables recognition that antiserum containing
anti-retroviral antibodies has been generated by exposure to the
non-infectious retrovirus-like particles by testing for antibodies
specific to the antigenic marker.
[0066] The vector can carry a codon-optimized retroviral, e.g., gag
gene, along with a heterologous promoter and transcription
terminator. A recombinant vector can also contain nucleic acid
sequences of an autonomous parvovirus joined to at least one
heterologous nucleic acid sequence. These autonomous parvovirus
nucleic acid sequences can comprise functional left and right end
inverted terminal repeats, so that heterolegous nucleic acid
sequence is located between and operably linked to parvovirus
nucleic acid sequences comprising left and right inverted terminal
repeats, and said vector is in a non-integrating form when
transferred into a cell.
[0067] In general, recombinant retroviruses carrying a vector
construct capable of preventing, inhibiting, stabilizing or
reversing infectious, cancerous or auto-immune diseases are
desirable. More specifically, the recombinant retroviruses of the
present invention are useful for inducing a specific immune
response to an antigen or a pathogenic antigen; essentially
inhibiting a function of a pathogenic agent, such as a virus; and
optionally inhibiting the interaction of an agent with a host cell
receptor.
[0068] This vaccine can comprise mixtures of at least 1 to about
1,000,000 different recombinant viruses each expressing a different
HIV env other viral gene variant or a portion thereof. Such genes
can contain both constant and variable regions. These genes and
their sequences are readily available from commercial and public
nucleic acid and amino acid sequence databases and depositories
like GenBank or ATCC, the content of which is incorporated herein
by way of reference. Even greater variation can be obtained by
substitutions or insertions of at least one nucleotide or one or
more amino acid residues, e.g., 1-25 amino acids. For example, the
viral proteins are sequence variants of the GP 120 protein of HIV
which differ from each other in terms of the amino acid sequence in
the area of the V2-loop and/or the V3-loop, preferably both the V2-
and V3-loop.
[0069] Without limiting to above embodiment the oral composition of
the invention contemplates a modified form of gpl20/160 of HIV-1,
said modified form of gp120/160 having a V3 loop disposed thereon,
said V3 loop being immunodampened so as to substantially redirect
an immune response away from the V3 loop on the modified form of
gp120/160 and toward a different part of said modified form of
gp120/160. More generally the invention cotemplates a modified form
of a native antigen of a pathogen, the native antigen having
disposed at a position thereon an immunodominant epitope comprising
a plurality of amino acids, said modified form of the native
antigen having a modified epitope at the position of the
immunodominant epitope of the native antigen, said modified epitope
having been immunodampened so as to substantially redirect an
immune response away from the modified epitope and toward a
different part of said modified form of the native antigen.
[0070] A genetically engineered cell line is contemplated
comprising an nucleic acid sequence encoding an HIV env protein
which is then cultured to produce unclipped HIV env protein, which
is recovered from the cell culture and prepared according to the
process of the invention. Alternatively, oligomeric and
glycosylated ectodomains of the surface protein gp160 of HIV as
well as the native protein domains themselves are obtained,
especially native ectodomains of the env glycoprotein of HIV whose
monomers exhibit an electrophoretic mobility of approximately 140
kD. A nucleotide sequence coding for a recognition sequence for
protein-splitting enzymes is inserted at a suitable site into the
gene coding for the precursor protein of the protein domain to be
obtained. After expression of the gene mutant in eukaryotic cells a
digestion with a suitable enzyme is carried out and the protein
domain to be obtained is subsequently purified.
[0071] The composition can also comprise the unprocessed
polyprotein initially translated from the genome of a virus and
which will contain epitopic configurations that are not retained in
the processed proteins. The composition can also contain cryptic
epitopes that are not usually "seen" by immune cells in their
natural presentation process. The present composition also
contemplates a recombinant protein encoding at least two antigenic
epitopes joined by flexible hinge domains. In this way the
invention is useful as a single vaccine composition effective
against diverse infectious agents since the subject proteins can
have antigenic epitopes from different infectious agents. Without
limitation the composition can contain mimotopes, e.g., a peptide
for therapeutic or prophylactic treatment of HIV infection
comprising an amino acid sequence or nucleic acid sequence
mimicking an antigen conformational epitope of HIV.
[0072] As another example a vaccine can comprise a complex of HIV
gp120 envelope protein covalently bonded to CD4 or to succinyl
concanvalin A or other lectin or hapten. In yet another embodiment
the vaccine preparation of the present invention does not contain
envelope proteins but contains essentially all other HIV proteins.
Such a preparation is either recombinant or prepared by routine
standard means by cleaving off envelope proteins and purifying env
stripped virions. Thus a non-infectious immunotherapeutic
containing retroviral particles devoid of outer envelope proteins
or containing selected antigens isolated from a retrovirus is
provided. Once this preparation is obtained one can easily make
vaccine preparation according to the preferred scope of the
invention. Such a preparation is equally suitable to produce
antibodies for passive immunotherapy, alone or in conjunction with
active immunotherapy, in individuals infected with a retrovirus,
including HIV.
[0073] Within the spirit of the invention one can construct a
peptide capable of eliciting in a mammal, a neutralizing immune
response against a pathogen comprising the steps of: generating a
neutralizing antibody specific for an epitope of an antigen of said
pathogen, wherein said antigen is a protein or polypeptide;
generating an anti-idiotypic antibody specific for said
neutralizing antibody; comparing amino acid sequences of said
anti-idiotypic antibody and said epitope; identifying an amino acid
sequence having at least 6 amino acids of a complementarity
determining region of said anti-idiotypic antibody that corresponds
to an amino acid sequence of said epitope; synthesizing a peptide
which contains said amino acid sequence of said anti-idiotypic
antibody that corresponds to an amino acid sequence of said
epitope; and administering such a peptide orally. One of specific
examples of a peptide eliciting a cytotoxic T lymphocyte response
and a high titer neutralizing antibody response is for example a
peptide having the following amino acid sequence
KQIINMWQEVGKAMYAPPISGQIRRIHIGPGRAFYTTKN which is formulated and
administered orally according to the spirit of this invention.
[0074] While advantages of above preparation are readily
appreciated one can nevertheless obtain an equally suitable
composition containing crude gp120 sequentially using ion exchange
chromatography, hydrophobic-interaction chromatography, and size
exclusion filtration, collecting at each step a fraction that
exhibits specific binding affinity for CD4 peptide. The process can
be carried out in the absence of any affinity purification steps or
any steps (such as reverse-phase HPLC) that use contact protein
with organic solvents. The product obtained by this method is a
purified, full-length, recombinant or native HIV gp120 glycoprotein
having protein/protein interaction properties substantially
identical to original gp120 as presented on an HIV virion,
including binding affinity for CD4 and binding affinity for at
least one antibody capable of neutralizing HIV infectivity.
[0075] Without limiting to examples cited supra, synthetic peptides
are provided which are advantageous vaccines against HIV. Such
peptides comprise an amino acid sequence of a T-cell epitope of the
gag protein of HIV, specifically p24 linked directly to an amino
acid sequence of a B-cell epitope of the V3 loop protein of an HIV
isolate and containing the sequence GPGR, and/or the gp4l
containing the sequence ELKDWA. Multimeric forms of the tandem
synthetic peptides are equally suitable. Another such peptide can
for example have a sequence
NH2-lysine-arginine-tryptophan-isoleucine-isoleucine-leucine-glycine-leuc-
ine-asparagine-lysine-soleucine-valine-arginine-methionine-tyrosine-cystei-
ne-COOH, which is derived from the gag p24 protein of HIV (i.e. one
of the internal core proteins) between residues 263 and 277. Other
virus-derived peptides which interact specifically with HLA
molecules are equally suitable.
[0076] Promoter can be CMV promoter, SV40 promoter, a PGK promoter,
vaccinia virus late or early promoter, or TK promoter. The vaccine
can further comprise a gene or a gene fragment encoding an
unrelated peptide, a polypeptide or a protein belonging to HIV or
other virus. A protein can comprise Nef and/or Tat of HIV and an
immunostimulatory CpG oligonucleotide. Without limitation an anti
HIV vaccine can comprise the entire or part of the Tat HIV 1
protein. The invention can also comprise polyepitopic proteinic
fragments of the HIV Nef protein, a method for the production and
use thereof.
[0077] The vector can also contain polyadenylation signals such as
a SV40 polyadenylation signal, a TK polyadenylation signals or an
HBV polyadenylation signal. A composition can also contain a
post-transcriptional RNA nucleocytoplasmic transport element
(NCTE), designated derived from a intracisternal A particle (IAP).
The IAP is inserted in a murine osteocalcinrelated gene (ORG)
between its promoter and ORG's coding region. Attenuated HIV hybrid
virus useful as a vaccine can equally contain a
post-transcriptional regulatory element that can function as an RNA
nucleo-cytoplasmic transport element. Additionally vector may carry
a gene or fragment coding for an interleukin, TNF, GM/CSF, a
nonretroviral viral antigen, e.g. gH, gD, gB or gL or a homologue
thereof, pertussis toxin, and/or a cancer antigen. Such a viral
vector may comprise a recombinant chimeric nucleic acid which is
derived from a nucleic acid encoding a fusion partner selected from
the group consisting of IL-1, IL-2, IL-4, IL-6, MART-1, gp 10,
tyrosinase, bcl-1, bcl-2, c-myc, int-2, hst-1, ras, p53,
prostate-specific membrane antigen, papilloma virus protein L1,
protein kinase C or G proteins.
[0078] A fusion polypeptide can also comprise a chemokine and
either a tumor or viral antigen which is administered as either a
protein or nucleic acid vaccine to elicit an immune response
effective in treating cancer or effective in treating or preventing
HIV infection. Also contemplated is a viral regulation protein or a
viral regulation protein along with alpha interferon or the alpha
interferon fragment which is carboxymethylated.
[0079] A live recombinant vaccine for HIV can be constructed using
an attenuated strain of Salmonella engineered to surface express
specific HIV proteins. Two recombinant plasmids, containing the
Lpp-OmpA genes required for surface exposure, are followed by the
genes for the HIV proteins, reverse transcriptase or
transactivating protein (Tat). These plasmids are electroporated
into an attenuated strain of Salmonella and antigen expression is
verified. Such vaccine is then given orally.
[0080] In another embodiment a polynucleotide comprising portions
of the genomes of caprine arthritis-encephalitis virus and HIV
produces a chimeric retrovirus, which is then delivered in a
pharmaceutically acceptable carrier.
[0081] Other recombinant chimeric viruses are equally suitable
including human rhinovirus 14 into which chimeric regions derived
from influenza HA, poliovirus or HIV are incorporated. Chimeric
human rhinoviruses are particularly advantageous as they are only
mildly pathogenic, have numerous potential serotypes and can elicit
significant mucosal and serum immunological response.
[0082] In yet another embodiment a nucleotide sequence encoding the
HIV regulatory protein NEF, REV or TAT or an immunologically active
fragment thereof is inserted into a vector comprising papilloma
virus nucleotide sequences necessary and sufficient for long-term
persistence. The resulting vectors are self-replicating and have a
high copy number.
[0083] More simple form of vaccine is also feasible having for
example essentially pure form of a hydrophilic peptide consisting
essentially of an amino acid sequence of about 9 to 35 units in
length and corresponding to at least one antigenic determinant of
the envelope glycoprotein of HIV recognized by immunocompetent
cells, e.g., B lymphocytes, said peptide, when optionally
covalently linked to a carrier molecule is administered orally
after prepared by the process of the invention.
[0084] In case of specifically anti-tumor type vaccine the
composition of the invention in addition to denatured tumor cells
and lysates thereof can also be enriched with recombinant or
naturally derived tumor antigens like MAGE-1, MAGE-3, MEL-1 and
peptide fragments thereof; human chorionic gonadotropin and peptide
fragments thereof; carcinoembryonic antigen and peptide fragments
thereof, alpha fetoprotein and peptide fragments thereof;
pancreatic oncofetal antigen and peptide fragments thereof; MUC-1
and peptide fragments thereof, CA 125, CA 15-3, CA 19-9, CA 549, CA
195 and peptide fragments thereof; prostate-specific antigens and
peptide fragments thereof; prostate-specific membrane antigen and
peptide fragments thereof; squamous cell carcinoma antigen and
peptide fragments thereof; ovarian cancer antigen and peptide
fragments thereof; pancreas cancer associated antigen and peptide
fragments thereof; Her1/neu and peptide fragments thereof; gp-100
and peptide fragments thereof; mutant K-ras proteins and peptide
fragments thereof; mutant p53 and peptide fragments thereof;
truncated epidermal growth factor receptor, chimeric protein
p210BCR-ABL and mixtures thereof.
[0085] Thermally denatured antigen of the invention is prepared by
heating antigen under neutral, acidic or alkaline conditions. As
shown later in examples, such a denaturation treatment results in
partial or complete inactivation of pathogen's infectious capacity.
The denatured antigen thus obtained can contain partially
disinfected pathogen. However, such a pathogen is essentially
non-viable, i.e., incapable of replicating itself more than once.
For example, a replication-defective HIV particle can be
pseudotyped with vesicular stomatitis virus G protein (VSV-G). The
pol gene of the HIV genome in the particle is modifed to inactivate
the pol reverse transcriptase and protease activity. This
pseudotyped HIV particle can infect many cell types, including
human and simian cells, and only undergoes one round of
replication.
[0086] For disintegrating for example antigens such as nucleic
acids in a biologically active proteinaceous material that
preserves its biological integrity one can admix a photodynamic or
photoactivatable substance, e.g., phenothiazine or methoxypsoralen,
and expose to activating light like laser beam light or ultraviolet
light or halogen light so that the nucleic acid content is
reduced.
[0087] Reduction or process of "reducing" as defined hereinafter
comprises a process including rendering, cooking, drying,
dehydrating, digesting, evaporating, pulverizing, sonicating,
protein concentrating, breaking into small pieces, reducing to
powder or granules, blasting, enzyme digestion, dialysis,
ultrafiltration, separating by gel migration, and ion exchange
chromatography.
[0088] While the denaturation of the antigen can sometimes result
from reducing process for the purposes of this disclosure the
process of denaturing includes heating, treating with a detergent,
oxidizing, aldehyde treatment, subjecting to extreme pH conditions
like acid or alkaline treatment, the preferred means are however
the thermal denaturation.
[0089] Although the conditions for the thermal denaturation may
vary depending on the heating temperature and period, heating for
longer than 10 min at a temperature higher than 60.degree. C. is
usually adopted. Furthermore, heating for longer than 15 min at a
temperature higher than 65.degree. C. is preferable, and an
autoclave is even more preferable, e.g., heating for about 20
minutes or more at 110.degree. C. or higher.
[0090] Thermal denaturation can be accompanied by acid and base
treatments. To provide acidic conditions, organic or inorganic acid
solutions are useful; e.g., citric, acetic, carbonic acid, or
hydrochloric acids. The pH range is preferably below 5.5 and more
preferably between 1.0 and 4.5. To provide alkaline conditions,
organic or inorganic alkali solutions are useful; e.g., sodium
hydroxide, potassium hydroxide, and triethanolamine. The pH range
is preferably above 8.5 and more preferably between 9.5 and
12.0.
[0091] The typical thermal denaturation conditions can be also
accompanied by pH conditions between 2.0 and 4.5 or between 10.0
and 12.0, at a temperature between 100 and 120.degree. C., for a
period between 10 and 15 minutes. In another embodiment, the
thermal-denaturation conditions are heating antigen in an inorganic
or organic acid solution for longer than 15 min or preferably for
longer than 20 min at above 65.degree. C. or more preferably at
about 100.degree. C., or heating in an autoclave for longer than 30
min or preferably for 2 to 10 h at about 110.degree. C., or for
longer than 15 min or preferably for 20 min or longer at about
120.degree. C.
[0092] Products containing thermally denatured antigen (e.g., soup)
can also be prepared by hot-water extraction of raw materials
containing antigen, e.g., cartilage of joints of sick animals.
[0093] The present inventor considers that the denaturation
treatments by heat or with the agents reduces the molecular weight
of antigen and destroys its steric molecular structure, and that
denatured antigen having the destructed helical structure is safer
causing less side effects than does native antigen. According to
another embodiment the three-dimensional structure of the antigen
is essentially intact.
[0094] It is not important that denaturation step follows
immediately the reduction step. For example, reduced antigen and
tissue can be formulated in the final product, e.g., functional
food-like meat products and fish products, dairy products like milk
or cheese, beverages like juice, milk, and tea, and liquid or solid
drugs and only then pasteurized or denatured.
[0095] Liquid compositions can contain conventional additives such
as suspending agents, for example sorbitol syrup, methyl cellulose,
glucose/sugar syrup, gelatin, hydroxymethylcellulose,
carboxymethylcellulose, aluminium stearate gel or hydrogenated
edible fats; emulsifying agents, for example lecithin, sorbitan
monooleate or acacia; non-aqueous vehicles, which can include
edible oils, for example vegetable oils such as arachis oil, almond
oil, fractionated coconut oil, fish-liver oils, oily esters such as
polysorbate 80, propylene glycol, or ethyl alcohol; and
preservatives, for example methyl or propyl p-hydroxybenzoates or
sorbic acid. Liquid compositions may conveniently be encapsulated
in, for example, gelatin to give a product in dosage unit form.
[0096] The reduced and denatured antigen can be eaten as foods or
as a food additive with or without enrichment with various
nutrients or dispersion in beverages to treat and prevent pathogen
infection. After adding the appropriate carriers and additives
according to the conventional practices, the functional foods may
be formed in shapes and forms suitable for eating (e.g., granule,
grain, tablet, capsule, and paste), added to various solid foods,
e.g., such meat products as hams and sausages, fish products,
candies, chocolate, candy bars, chewing gums, flour, flakes, bread,
milk powder, breast-milk formulas, and butter, and various liquid
or semi-solid foods (potable water, jam, juice, milk, wine, beer,
purees, infant foods, ice-cream, and soft drinks). For example,
denatured composition and natural juice can be blend at desired
ratio, e.g., 0.5 mg to 200 ml, sterilized conventionally and
packaged aseptically to obtain a juice product.
[0097] The dosage of denatured antigen formulated in various
functional foods may properly be adjusted depending on the age,
body weight, severity, degrees of the disease of the patient, and
shape of the food. The dose of the antigen may range between 0.0005
microgram/kg and 15 g/kg of body weight/day. Intake of more or less
than those dosages, however, is harmless, since denatured antigen
has no harmful effect on living bodies, even if a large amount is
taken.
[0098] The active component of denatured antigen can be blended
with solid or liquid pharmaceutical carriers prepared in various
pharmaceutical forms and administered. Dosage forms can include
pharmaceutically acceptable carriers and adjuvants which are known
to those of skill in the art.
[0099] An exemplary adjuvant composition of an oil-in-water
submicron emulsion may consist essentially of about 0.5 to 50% of a
first component of an oil, about 0.1 to 10% of a second component
of an emulsifier, wherein the emulsifier is a phospholipid compound
or a mixture of phospholipids selected from the group consisting of
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,
sphingomyelin and cardiolipin, about 0.05 to 5% of a non-ionic
surfactant, about 0.00001, to 10% of an immunogen of the invention,
and an aqueous continuous phase. This submicron emulsion can have a
mean droplet size in the range of between about 0.03 and 0.5
micrometer.
[0100] Another exemplary adjuvant can contain one or more
adjuvants/vehicles like polyoxyethylene sorbitan monoesters,
polyoxyethylene castor oil, caprylic/capric acid glycerides and
gangliosides in an amount of about 0.01 to 45% (v/v) calculated on
the total volume of the preparation.
[0101] Other typical carriers and adjuvants include, for example,
ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, chitosan, buffer substances
such as phosphates, glycine, sorbic acid, potassium sorbate,
partial glyceride mixtures of saturated vegetable fatty acids, one
or more saponin-lipophile conjugates, desacylsaponin, triterpene
saponins, saponarin, sarmentocymarin, sapogenins, sarmentogenin,
sarsasapogenin, sarverogenin,
N-palmitoyl-S-2,3(bispalmitoyloxy)-propyl-cysteinyl-seryl-serine,
an unsaturated turpin hydrocarbon, like squalene or squalane, a
polyoxypropylene-polyoxyethylene block copolymer, anionic lipids
like salts of lauric and oleic acids, lauric and oleic acids, acid
esters of lauryl and cetyl alcohol, and sulfonates, lectins,
estrogenic compounds, a peptide to which has been attached a
hydrophobic tail, said peptide being adsorbed to viral particles
comprising intact virus surface antigen attached via said
hydrophobic tail, a synthetic peptide carrier which may constitute
a T cell epitope, e.g., one derived from the which corresponds to
positions 437-453 of E. coli hsp65 (GroEL), or an analog thereof,
cyclic peptides containing as constituting strand(s) one or two
amino acid sequences selected from among amino acid sequences
Glu-Ala-Asp-Asp-Arg and/or Ser-Gln-Lys-Glu-Gly, peptide having the
amino acid sequence
X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys-Gly-Glu-Ile-Lys-A-
sn-Cys-Ser-Phe-Asn-Ile-Ser-Thr-Ser-Y wherein X is either a hydrogen
atom of the amino terminal NH2 group of said peptide or an
additional amino acid selected to facilitate coupling of said
peptide to a carrier and Y is selected from the group consisting of
an amino group, hydroxy group, cysteine residue, cysteine residue
followed by an amino group and cysteine residue followed by a
hydroxy group, hemagglutinin protein, BCG, diphtheria, tetanus,
whole cell pertussis, polio, hepatitis B, hemophilus influenza,
measles, mumps and rubella immunogens, or any other viral, fungal,
bacterial, protozoan or parasite protein/immunogen that in
combination can elicit desired immune response, hydroxylated lower
alkyls, dimethyl sulfoxide, urea, water, salts or electrolytes such
as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances and polyethylene glycol. Adjuvants for topical or gel
base forms of the compounds and compositions of this invention
include, but are not limited to, sodium carboxymethylcellulose,
polyacrylates, waxes, polyoxyethylene-polyoxypropylene-block
polymers, polyethylene glycol, propylene glycol and wool fat.
[0102] A pharmaceutically acceptable salt is a salt that retains
the biological effectiveness and properties of the active
ingredient of the composition and which is not biologically or
otherwise undesirable. The present composition can be provided in
form of a salt. Salts can be derived from acids or bases. The acid
addition salts can be derived from inorganic acids, such as
hydrochloric acid, hydrobromic acid, sulfuric acid (giving the
sulfate and bisulfate salts), nitric acid, phosphoric acid and the
like, and organic acids such as acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, salicylic acid, p-toluenesulfonic acid,
and the like. The base addition salts can be derived from inorganic
bases, and include sodium, potassium, lithium, ammonium, calcium,
magnesium salts, and the like. Salts derived from organic bases
include those formed from primary, secondary and tertiary amines,
substituted amines including naturally-occurring substituted
amines, and cyclic amines, including isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine,
ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine,
arginine, histidine, caffeine, procaine, hydrabamine, choline,
betaine, ethylenediamine, glucosamine, N-alkylglucamines,
theobromine, purines, piperazine, piperidine, N-ethylpiperidine,
and the like. Preferred organic bases are isopropylamine,
diethylamine, ethanolamine, piperidine, tromethamine, and
choline.
[0103] Compositions for topical administration include ointments,
creams, gels, lotions, shampoos, paints, powders (including spray
powders), pessaries, tampons, sprays, dips, aerosols, pour-ons and
drops. Topical administration includes administration to the skin
or mucosa, including surfaces of the lung and eye. Compositions for
topical administration, including those for inhalation, may be
prepared as a dry powder which may be pressurized or
non-pressurized. In non-pressurized powder compositions, the active
ingredient in finely divided form may be used in admixture with a
larger-sized pharmaceutically-acceptable inert carrier comprising
particles having a size, for example, of up to 100 micrometers in
diameter. Suitable inert carriers include sugars such as lactose.
Desirably, at least 95% by weight of the particles of the active
ingredient have an effective particle size in the range of 0.001 to
10 micrometers. The active ingredient can, for example, be
formulated in a hydrophilic or hydrophobic base as appropriate.
[0104] The aerosol type agent can be prepared generally by
providing a sterilized solution or suspension containing the
effective ingredient and then adding a propellant thereto. In
preparing the solution or suspension, there can be used all
diluents commonly used in the art, such as those mentioned with
respect to the injection. As to the propellant, there can be used
all propellants commonly used in the art, such as liquefied gas
propellants for example, chlorofluorocarbons (e.g., F1on 12 or F1on
123), compressed gas propellants (e.g., nitrogen gas and carbon
dioxide) and the like. The aerosol type agent may further contain a
solubilizing agent, a buffer solution, etc., which are used
commonly and, if necessary, the aerosol type agent may contain a
coloring agent, a preservative, a perfume, a flavoring agent, a
sweetening agent, etc.
[0105] Examples of oral pharmaceutical forms are, e.g., solid drugs
as tablet, granule, powder, and capsule, such liquid ones as
solution, suspension, and emulsion, and lyophilized one, inhalants,
skin patches, bolus pumps, controlled release devices,
suppositories, and trans-intestinal liquid. These drugs can be
prepared by conventional pharmaceutical practices. Examples of the
above-described pharmaceutical carriers are glucose, lactose,
sucrose, starch, mannitol, dextrin, fatty-acid glyceride,
polyethylene glycol, hydroxyethyl starch, ethylene glycol,
polyoxyethylene sorbitan fatty-acid ester, amino acids, albumin,
water, and physiological saline. Furthermore, such conventional
additives as stabilizer, smoother, humectant, emulsifier, and
caking agent can be added. Tablets and capsules according to the
invention can, if desired, contain conventional ingredients such as
binding agents, for example syrup, acacia, gelatin, sorbitol,
tragacanth or polyvinyl-pyrollidone; fillers, for example lactose,
sugar, maize-starch, calcium phosphate, sorbitol or glycine;
lubricants, for example magnesium stearate, talc, polyethylene
glycol or silica; disintegrants, for example potato starch; or
acceptable wetting agents such as sodium lauryl sulphate. Tablets
can be coated according to methods well known in the art.
[0106] In shaping the external preparation like tablets and pills,
there can be used various materials known in the art. As the base,
for example, there can be used at least one base selected from
various oily bases or various water-soluble bases. Specific
examples of the base are oils and fats such as peanut oil, sesame
oil, soybean oil, safflower oil, avocado oil, sunflower oil, corn
oil, rape seed oil, castor oil, camellia oil, coconut oil, olive
oil, poppy seed oil, cacao oil, beef tallow, lard, and the like;
modified oils and fats obtained by subjecting the above-mentioned
oils and fats to chemical treatments (e.g. hydrogenation); mineral
oils such as petrolatum, paraffin, silicone oil, squalane and the
like; higher fatty acid esters, higher aliphatic alcohols and waxes
such as isopropyl myristate, N-butyl myristate, isopropyl
linoleate, propyl ricinoleate, isopropyl ricinoleate, isobutyl
ricinoleate, heptyl ricinoleate, diethyl sebacate, diisopropyl
adipate, cetyl alcohol, stearyl alcohol, bleached bees wax,
spermaceti, Japan wax, hydrous lanolin, camauba wax, shellac wax
and the like; higher fatty acids such as stearic acid, oleic acid,
palmitic acid and the like; mixtures of mono-, di- and
tri-glycerides of saturated or unsaturated fatty acids of 12-18
carbon atoms; polyhydric alcohols such as ethylene glycol,
polyethylene glycol, propylene glycol, polypropylene glycol,
glycerin, batyl alcohol, pentaerythritol, sorbitol, mannitol and
the like; gummy substances such as acacia, gum benzoin, guaiacum,
tragacanth gum and the like; natural water-soluble polymers such as
gelatin, starch, casein, dextrin, pectin, sodium pectate, sodium
alginate, methyl cellulose, ethyl cellulose, carboxymethyl
cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,
nitrocellulose, crystalline cellulose and the like; synthetic
water-soluble polymers such as polyvinyl alcohol, polyvinyl methyl
ether, polyvinylpyrrolidone, sodium polyacrylate, carboxyvinyl
polymer, polyethyleneimine and the like; nonionic, anionic or
cationic or amphoteric surfactants; ethanol; isopropanol; and
water. In the present invention, these bases can be used singly or
in admixture of two or more.
[0107] Generally, according to the invention any infected tissue as
a starting material is adequate and suitable. The preferred
embodiment of the invention is blood. Blood is collected,
preferably from donors already infected with the pathogen.
According to this embodiment, the blood can be held with
anticoagulants such as sodium citrate or EDTA to prevent clotting.
Prevention of clotting is not essential to the invention.
Typically, the whole blood is separated, preferably by
centrifugation, although any other separation method may be used,
into two parts, the cellular solid material (red cells, white
cells, platelets, and other circulating cells or precursors
thereof) and liquid form like plasma or serum. As used herein the
term "plasma" shall include the serum and plasma portion of blood
as well as any of the protein and components which may be further
purified therefrom. Plasma and/or the purified components of
plasma, may then be further concentrated (by membrane filtration).
The blood is next dried to form a beige/brown powdery substance.
When this powder is washed and dried again it results in white
crystalline powder. The resulting powdery substance can have a
particle size of about 0.5 to about 30 microns or higher.
Optionally, the powder can be further compacted or compressed
(around 1200 to 1400 psi) to form granules and screened or
otherwise separated by size to increase homogeneity. The resulting
granulated particle size is at least about 50 microns. Preferably
the size is greater than about 100 microns in diameter. The
resulting granular substance can then be combined with other
ingredients for any desired therapeutic regimen or may be blended
with an ingredient and the blend granulated. The granulated blended
composition preferably comprises from greater than 0 to about 50%
by weight of the base product.
[0108] The composition of the invention can be admixed with a
natural polymer like starch, dextran, dextrin, and maltodextrin or
a hydrolysate of a natural polymer, or a mixture thereof. Natural
polymer can be a cross-linked polysaccharide, a cross-linked
oligosaccharide, a derivative or hydrolysate of a cross-linked
polysaccharidec or a cross-linked oligosaccharide, and a mixture
thereof. The core containing the composition can be uncoated, or is
partially or completely coated with no more than one layer, the
layer comprising a lipid compound covalently bonded to the core, or
an amphiphilic compound.
[0109] In addition to granulation, many methods, such as pelleting,
wet or dry agglomeration, prilling, and fluid-bed drying, can be
used to modify as desired the particle size and density of dried
blood or plasma and/or its component proteins and are included
within the scope of the invention.
[0110] According to another embodiment of the present invention,
the blood composition is obtained from animal sources, granulated
and fed with other feed ingredients to domestic animals, which need
intervention against pathogen infection. Any animal in which it is
desirable to prevent or treat infection, can be fed the composition
according to the invention. These animals include but are not
limited to cats, dogs, cows, sheep, pigs, chicken, turkeys, ducks,
horses, donkeys, camels, goats or aquaculture. Wild animals in need
of treatment or prevention are also contemplated as targets of
intervention.
[0111] The composition is obtained by collecting blood from
pathogen-infected animals. Infection can be natural or deliberate
to create starting material for the composition. Naturally infected
blood, which is traditionally discarded, can be used for
preparation of the compositions and implementation of methods of
the invention.
[0112] In further aspects, methods are provided for administering
any of the above-described composition, for a prophylactic or
therapeutic effect. For example, within one aspect, the present
invention provides methods of stimulating or inhibiting an immune
response to an antigen or modified form thereof being capable of
modulating an immune response within an animal.
[0113] In another embodiment, the cells of a healthy animal are
removed, infected ex vivo by a pathogen, made into a composition
and returned to the animal via mucosal surface, e.g., orally. In
yet another embodiment, tissues and pathogen from a sick animal are
processed according to this instant process and fed back to the
same animal.
[0114] The specific therapeutically effective doses for any
particular patient will depend upon a variety of factors including
the disorder being treated and the severity of the disorder;
activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coincidental with the specific compound employed; and like
factors well known in the medical arts. For example, it is well
within the skill of the art to start doses of the compound at
levels lower than required to achieve the desired therapeutic
effect and to gradually increase the dosage until the desired
effect is achieved. This is a routine clinical procedure that does
not require the undue experimentation. Generally, the total daily
dose of the compounds of this invention administered to a human or
lower animal may range from about 0.001 to about 100 mg/kg of
patients body mass/day. For purposes of oral administration, more
preferable doses may be in the range of from about 0.005 to about
30 mg/kg/day. If desired, the effective daily dose may be divided
into multiple doses for purposes of administration; consequently,
single dose compositions may contain such amounts or submultiples
thereof to make up the daily dose. If necessary, the composition
can be given in less frequent regimen, e.g., once every 3 days,
once weekly, once monthly, once every 3 months, or once every 6
months as needed. Doses given once a year or less frequently as
required by exigencies of the therapy or patient's condition are
also contemplated as equally suitable. In general, the therapy can
be administered as often as necessary and for the duration of time
deemed necessary. An effective amount means that amount necessary
to delay the onset of, inhibit the progression of, halt altogether
the onset or progression of or diagnose the particular condition
being treated. In general, an effective amount for treating for
example cancer will be that amount required to inhibit mammalian
cancer cell proliferation in-situ either directly or indirectly via
recruitment of immune cells. When administered to a subject,
effective amounts will depend, of course, on the particular
condition being treated; the severity of the condition; individual
patient parameters including age, physical condition, size and
weight; concurrent treatment; frequency of treatment; and the mode
of administration. These factors are well known to those of
ordinary skill in the art and can be addressed with no more than
routine experimentation. It is preferred generally that an optimal
dose be used, that is, the safest and the most potent dose
according to sound medical judgment.
[0115] Dosage may be adjusted appropriately to achieve desired drug
levels, locally or systemically. Generally, daily oral doses of
active compounds will be from about 0.0001 mg/kg per day to 1000
mg/kg per day. Preferably the systemic levels of the composition
are within the same range as shown effective in an in vitro and
animal models. It is expected that injectable doses in the range of
about 1 to 1000 mg/m.sup.2 per day will be effective and the
optimal dosage is determined by a routine pharmacokinetic,
dosage-scaling experimentation well within the skill of the
practitioner. In the event that the response in a subject is
insufficient at such doses, even higher doses (or effective higher
doses by a different, more localized delivery route) can be
employed to the extent that patient tolerance permits. Continuous
dosing over, for example 24 hours or multiple doses per day are
contemplated to achieve appropriate systemic levels of
compounds.
[0116] The preventive and therapeutic methods described herein can
be used alone or in conjunction with additional therapy known to
those skilled in the art for the treatment of a given disease or
condition. The pharmaceutical composition of the present invention
can also comprise other active agents or drugs. In yet another
embodiment, the composition can be administered in combination with
other drugs or pharmaceutically active ingredients including but
not limited to vitamins, anthracycline, i.e., doxorubicin,
4'-epi-doxorubicin, 4- or 4'-deoxydoxorubicin, etoposide,
epothilone A-C, N-bis(2-chloroethyl)-4-hydroxyaniline,
4-hydroxycyclophosphamide, vindesine, vinblastine, vincristine,
terfenadine, terbutaline, fenoterol, salbutamol, muscarine,
oxyphenbutazone, salicylic acid, p-aminosalicylic acid,
5-fluorouracil, methotrexate, diclofenac, flufenamic acid,
4-methylaminophenazone, theophylline, nifedipine, mitomycin C,
mitoxantrone, camptothecin and camptothecin derivatives,
N-[4-(9-acridinylamino)-3-methoxy-phenyl]-metha- nsulfonamide
("m-AMSA"), taxol or other taxanes, nocodaxol, colchicine,
fexofenadine, cyclophosphamide, rachelmycin, cisplatin, melphalan,
bleomycin, nitrogen mustard gas, phosphoramide mustard gas,
verrucarin A, neocarcinostatin, calicheamicin, dynemicin,
esperamicin A, quercetin, genistein, erbstatin, tyrphostin,
rohitukin derivative, retinoleic acid, butyric acid, phorbol ester,
dimethyl sulfoxide, aclacinomycin, progesterone, buserelin,
tamoxifen, mifepristone, onapristone,
N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide,
pyridinyloxazol-2-one, quinolyl- or isoquinolyloxazol-2-one,
staurosporin, ethanolamine, verapamil, forskolin,
1,9-dideoxyforskolin, quinine, quinidine, reserpine,
18-O-(3,5-dimethoxy-4-hydroxybenzoyl)-rese- rpate, lonidamine,
buthionine sulfoximine, diethyl dithiocarbamate, cyclosporin A,
rapamycin, azathioprine, chlorambucil, hydroxycrotonamide
derivative 2, leflunomide, 15-deoxyspergualine, FK 506, ibuprofen,
indomethacin, aspirin, sulfasalazine, penicillamine, chloroquine,
dexamethasone, prednisolone, mefonamidic acid, acemannan,
acetaminophen or paracetamol, 4-aminophenazone, chloroquine,
amodiaquin, hydroxychloroquine, primaquine, quinacrine muskosine,
orciprenaline, isoprenaniline, thalidomide, linomide, amiloride,
p-nitrophenyl guanidine benzoate, 1,25-dihydroxy Vitamin D3,
biologically active Vitamin D3 derivatives, all trans-retinoic
acid, retinoic acid derivatives, retinol, retinol derivatives,
glucocorticoids, dehydroepiandrosterone (DHEA), DHEA-derivatives
like DHEA-sulfate (DHEA-S), 16-alpha-bromo-DHEA, 7-oxo-DHEA,
16-alpha-Br-DHEA-S, 7-oxo-DHEA-S or their derivatives in which one
or more hydroxyl, amino or imino groups have been additionally
substituted. Other drugs contemplated in this invention can be
readily glanced from public databases like
http://pharminfo.com/drugdb/db_mnu.htm- l; www.rxlist.com;
http://www.medicineinfo.com; www.phrma.org; www.aphanet.org;
www.pjbpubs.co.uk; www.drugbase.co.za; http://www.prescript.com; or
http://www.musclerelaxant-medications.com/in- dexl.htm incorporated
herein by way of reference. These active compounds belong to
various classes of drugs like antitumor agents, standard
cytostatics, antimetabolites, substances that intercalate DNA,
inhibitors of topoisomerases, tubulin inhibitors, alkylating
agents, compounds that inactivate ribosomes, tyrosine phosphokinase
inhibitors, differentiation inducers, hormones, hormone agonists or
hormone antagonists, substances which modify the pleiotropic
resistance to cytostatics, calmodulin inhibitors, protein kinase C
inhibitors, G protein receptor antagonists, cell cycle-related
molecule antagonists, P-glycoprotein inhibitors, modulators of
mitochondrially bound hexokinase, inhibitors of
gamma-glutamylcysteine synthetase or of glutathione S-transferase,
inhibitors of superoxide dismutase, inhibitors of the
proliferation-associated protein in the cell nucleus of dividing
cells, substances which exert immunosuppressant effects, standard
immunosuppressants, macrolides, nonsteroidal antiinflammatory
substances, antirheumatic drugs, steroids, antiinflammatory,
analgesic or antipyretic substances, antiviral drugs, antibiotics,
derivatives of an organic acid, nonacidic analgesics or
antiinflammatories, local anesthetics, medicinal herbs and plants,
antiarrhythmics, Ca.sup.++ antagonists, antihistaminics,
phosphodiesterase inhibitors, parasympathomimetics,
sympathomimetics or substances having inhibitory action on human
urokinases among many others.
[0117] The following examples serve to illustrate the invention and
are not intended to limit the invention in any way. Those skilled
in the art will recognize that there are several different
parameters which may be altered using routine experimentation and
which are intended to be within the scope of this invention.
EXAMPLES
Example 1
Carbon Dioxide Process
[0118] Whole blood or blood serum or plasma or cell culture medium
with pathogen-infected cells present in them are treated with
carbon dioxide (CO.sub.2) in a pressurized vessel and heated. Other
common gases like ozone, oxygen, nitric oxide (NO), liquid
nitrogen, and mixtures thereof can be equally used.
[0119] Under these conditions, CO.sub.2 reacts with the moisture in
the vessel to form a weak acid. The combination of acid and heat
causes the ingredients such as cells and proteins to form two
fractions, one a solid and the other a liquid. The solid fraction
is of interest for use in making instant composition. The liquid
fraction is also useful since it contains cell-free pathogen. To
separate the fractions, two different methods are equally suitable:
centrifugation and membrane separation. In centrifugation, the
mixture of the fractions is placed in a container and spun at a
high rate of speed so that the heavier solid fraction settled to
the bottom of the container. The liquid fraction is decanted off
and further processed. In membrane filtration, the mixture of the
fractions is pumped past or poured over a porous membrane. The
fractions are separated easily because the liquid fraction passes
through the pores of the membrane leaving a concentrated solid on
the surface of the filter. Both methods are equivalent in recovery
of the proteins and solid matter. The solid matter can be further
processed, e.g., autoclaved, made into a powder, and compounded
into granules or tablets. These steps can be in any order.
[0120] The additional advantage of gas treatment is provided by
explosion or bursting of the target antigen by gas bubbles into
smaller fragments, effectively reducing the initial size. The
reduced form is inherently the part of the body of antigen.
According to one of embodiments the reduced antigen keeps its
physical structure and shape such as their molecular and
supramolecular curve and cleft close to the initial
three-dimensional form as much as possible. According to another
embodiment the reduced form is at variation with the initial
structure. In yet another embodiment the original monodisperse
colloids and crystals of the composition are effectively converted
into hollow shapes pretty much as in "lost-wax" approach. These are
encased in the pharmaceutical carrier and formulated as an oral
pill. Upon digestion in the gut the carrier releases the original
parts or fragments of an antigen and presents it to the immune
system of a host when it is processed via usual cellular pathways,
e.g., antigen presentation via MHC Class 1 or 2 by specialized
mucosal (regular enterocyte, Peyer's patch cell or M cell) and/or
perimucosal dendritic cells residing in mesenteric lymph nodes or
in Peyer's patch and functioning as antigen presenting cells (APC).
The vaccine protein or polypeptide which is either natural or
recombinant can thus comprise, for example, the alpha-3 domain of a
MHC class I molecule, a membrane-anchoring structure and an
antigenic polypeptide determinant. This antigenic polypeptide
determinant can substitute either a portion of the alpha-1, alpha-2
or both the alpha-1 and alpha-2 domains of MHC class I molecule,
wherein said polypeptide vaccine is expressed in membrane-bound
form on the surface of a cell and wherein said antigenic
polypeptide determinant is capable of interaction with T-lymphocyte
receptor.
[0121] Equipment used for this and other reduction and denaturation
processes are commercially available. One skilled in the art can
easily select appropriate ones among Rotary Drums, Sectional
Coolers, (Drying and Cooling) Flash Dryers, Fluidized Bed Plants,
Turbo--Tray Dryers, Solidizer, Disc Dryers, Spirocorn, Belt Dryers,
Tray Dryers Simplicior and Favorit, Air Mix, Spray Mix, Mills and
Classifiers and order them from various manufacturers like Duske
Engineering, Inc. Fenwal, Baxter, among many others.
Example 2
Freeze-drying Process
[0122] The prior art method for freeze-drying or lyophilization is
well established, for example, in Remington's Pharmaceutical
Sciences, 18th Edition, Mack Publishing Company, Easton, Pa.,
U.S.A., or in U.S. Pat. No. 4,001,944.
[0123] This method uses a vacuum chamber and a condenser. The
aqueous product contained in bottles or tissue blocks are frozen in
an inert atmosphere at a temperature below its eutectic
temperature; the samples are supported on a refrigerated shelf in
the chamber. The chamber is evacuated usually below 0.1 Torr (100
microns of mercury pressure). The ice which is produced is then
sublimed from the product onto the condenser at a temperature below
that of the product. Finally, heat is introduced to the product by
warming the shelf to provide energy for sublimation while keeping
the product below its eutectic temperature.
1TABLE 1 Typical amino acid profile of blood-derived composition
(%) Crude protein 86 Arginine 3.6 Histidine 5.2 Isoleucine 0.9
Lysine 7.5 Methionine 1.0 Threonine 3.6 Tryptophan 1.0 Valine
7.5
Example 3
Spray Drying Process
[0124] The process described below is for whole blood but can be
easily carried out with any blood cell fraction or cell culture
instead. The process is preferably carried out in an assembly of
apparatuses comprising a container with indirect steam heating, a
container with direct steam heating and a feeding screw, a
centrifuge, a grinding equipment, an autoclave with a supply of
steam, a second centrifuge, an evaporation equipment, a spray
drier, a mixing vessel, and an optional second spray drier. These
apparatuses can be interconnected so that the process is automated
and involves limited operator involvement. For the sake of
convenience, the quantities given below are based on the processing
of 1 liter or 1 kg of raw blood. One kg of whole blood with a dry
matter content of about 16% by weight is fed into the first
container and heated to 30-40.degree. C. by introduction of steam
into the jacket of the container. After remaining in the container
for one hour, the blood is fed to a second container, where it is
heated to 95-98.degree. C. by blowing in steam. After the blood has
coagulated, it is fed to a centrifuge, where it is separated into a
water phase and a solid phase. The maturation process preceding the
coagulation takes place at a slightly raised temperature, and has
the effect of minimizing the loss of protein with the water phase
leaving the centrifuge, the protein content of this water phase is
preferably less than about 1% by weight. The major part of the
salts in the whole blood (including the anticoagulant citrate used
during blood collecting) is contained in the water phase, which can
be discarded. The solid phase leaving the centrifuge can be as low
as 0.15 kg dry matter and 0.225 kg water. Thus, the dry matter
content is approximately 40% by weight. The solid phase can be
diluted with water to a dry material content of about 25% by
weight, and then is fed to the grinding equipment. The grinding
serves to create the greatest possible surface area for the
subsequent hydrolysis process and prevents agglomeration of the
coagulated blood particles. After grinding, the solid phase is
transferred to the pressure container. The solid phase is heated in
the container by directly blowing in pressurized steam, and it is
hydrolyzed at a pressure of approximately 600-800 kPa (6-8 bar) and
a temperature of 120-170.degree. C. for about 10-60 minutes. During
hydrolysis, some of the amino acid links in the protein are broken,
which results in short-chain, water soluble peptides. After the
hydrolysis process, the hydrolyzed blood mass is fed to the
centrifuge or to an ultrafiltration equipment, in which the mass is
separated into a liquid phase containing soluble peptides, and a
solid phase containing non-hydrolyzed protein. The solid phase,
which contains about 40% by weight of dry matter, is diluted to a
solid content of about 25% by weight in the mixing vessel, and is
then spray-dried in the spray drier at appropriate temperature. In
a spray drying machine it is preferable that inlet temperature is
approximately 375 to 400.degree. F. and an outlet temperature from
the drier of 180-200.degree. F. This yields a blood powder as a
final product. The liquid phase, which contains less than 16% by
weight of dry matter, is evaporated in the evaporation equipment to
a dry matter content of 35-40% by weight. The concentrate is then
fed to the spray drier and spray-dried to a fine powder. The
mixture of two blood powders obtained by the spray-drying process
is light brown and slightly bitter tasting. It contains about 90%
of raw or total protein by weight; fat or lipids at about 0.3%;
ashes about 5%; iron about 360 mg/kg and water about 5%.
Spray-dried plasma powder is obtained by a similar process. The
obtained powder is able to pass through a 12 mesh screen and is
collected on a 30 mesh screen. In addition, spray dried animal
plasma is commercially available from several sources including
American Meat Protein Corporation product sold under the mark of
AP920 and can be added to instant powder as a "bulking" additive,
especially in case of a composition made out of cell cultures.
[0125] An optional granulation of powder is equally suitable and
desirable. Granulation has long been used to improve flowability of
fine hygroscopic powders and ease of handling. It is also expected
that granulation would beneficially affect the clinical efficacy,
beyond simply what is observed for similar amounts of powder. While
granulation step is well known to those of skill in the art a
typical compaction/granulation system for use in the present
invention consists of following. Normally, the powder leaves the
powder feed and advances through a screw wherein the powder is
pre-compressed and acerated. The pre-compressed powder then enters
a compaction chamber containing compaction rolls. Therein, a
hydraulic actuator regulates the amount of pressure exerted on the
rolls. From the compacting chamber, the compressed material enters
a grinding mill and thereafter passes through a screen wherein the
granules are screened to the desired size. The granular product of
appropriate particle size is then gathered. The "overs" are
recycled with the "fines" from the process through the recycle
system or may be recycled through the grinding mill wherein the
granulation process is repeated. The granular product is then
stored in moisture-resistant containers. Without being limited to
particular doses and ranges the representative chemical and other
properties of the composition include about 60-90% protein, about
5-9% moisture, about 5-20% ash, about 1-5% fat, iron, calcium,
chloride, phosphorous, potassium, and other ingredients inherently
present in original raw material. A typical amino acid assay of the
powder by acid hydrolysis and subsequent column chromatography
results in the following amino acid concentrations (grams per 100
grams of powder):
2TABLE 2 The composition of three representative batches (%) Batch
#1 Crude protein 50 Cystine 0.3 Methionine 0.9 Aspartic acid 11.2
Threonine 3.9 Serine 5.1 Glutamic acid 10.6 Proline 4.1 Glycine 5.1
Alanine 8.5 Valine 9.1 Isoleucine 1.0 Leucine 12.1 Phenylalanine
5.9 Histidine 6.2 Lysine 8.9 Arginine 3.7 Tryptophan 1.9 Batch #2
Crude protein 60 Alanine 4.2 Arginine 4.7 Aspartic Acid 7.9 Cystine
2.8 Glutamic Acid 11.7 Glycine 3.0 Histidine 2.8 Isoleucine 2.9
Leucine 7.8 Lysine 6.8 Methionine 0.7 Phenylalanine 4.6 Proline
12.8 Serine 4.7 Threonine 4.8 Tryptophan 1.4 Tyrosine 3.6 Valine
6.3 Batch #3 Crude Protein 78 Isoleucine 2.3 Lysine 7.4 Methionine
2.4 Threonine 4.2 Tryptophan 1.2
Example 4
Preparation of White Powder
[0126] A number of additional steps can be carried out to further
modify the composition as desired. For example, to obtain a product
having higher whiteness degree one can adopt the procedures well
known in the art as disclosed in Japanese patent JP1988000041437
and/or U.S. Pat. No. 4,262,022. Accordingly, whole blood, blood
before separation, erythrocyte, hemoglobin or a dried substance
thereof is diluted with an alcohol or an aqueous solution of an
alcohol (e.g., about 2.5 to 5 times dilution ratio of solid content
at about 40% alcohol concentration), adjusted to pH around 1.5 to
5.5, preferably 2 to 5, incorporated with 0.05 to 0.6 the solid
content weight of a chlorite and heated at 40 to 65.degree. C. to
discolor hemoglobin or hemoglobin-containing blood. Another process
for producing a decolorized edible material from blood is achieved
by hemolyzing a red blood corpuscle containing blood fraction; then
partially hydrolyzing the hemolyzed blood fraction enzymatically
with the proteinase of Bacillus licheniformis to a degree of
hydrolysis of at least 10, forming a hydrolyzate constituted of a
decolorized supernatant containing partially hydrolyzed protein
dissolved therein and a sludge; and thereafter deactivating the
proteinase and recovering the supernatant.
[0127] While these steps are suitable one skilled in the art can
simply proceed with a repeated washing steps with simple solvent
such as distilled water until the baked brownish powder becomes
white in appearance.
Example 5
Composition Other Than Powder
[0128] This invention also provides a process for preparing non
powdered product comprising: reacting muscle cells or blood with a
nitrosating agent and a reductant, at elevated temperatures, to
provide a cooked cured-meat-like product; stabilizing and/or
encapsulating and/or protecting the cooked cured-meat, to provide a
stabilized cooked cured-meat; and drying the stabilized cooked
cured-meat by spray-drying, drum-drying or freeze-drying
techniques. This invention also provides the combination of an
emulsion-type meat or fish product with a cooked cured-meat and a
nitrosating agent, in stabilized, encapsulated or protected form. A
composition containing water is also contemplated having dispersed
therein a nucleic acid or peptide or a plurality of active peptides
or oligonucleotides each of which consists essentially of 2 to
about 500 residues having a sequence that corresponds to a portion
of a conserved domain of an HIV protein or genome, said
composition, when used to immunize an animal, having the capacity
to induce cell-mediated immune activation as well as inducing
humoral immune reaction, i.e., antibodies, that immunoreact with
said corresponding native HIV.
Example 6
Sodium Hydroxide and Acid Hydrolysis by pH Adjustment
[0129] Bovine blood cells (10 g) are added to 90 mL of 8:1 (v/v)
distilled water/sodium hydroxide containing reductant(s) into which
a nitrosating agent is introduced. Reducing agents (ascorbic acid,
erythorbic acid, and/or ascorbyl palmitate) are added to the
reaction mixture at a heme in RBC to reductant mole ratio of 1:5,
1:10, or 1:20. Sodium nitrite, the nitrosating agent employed, is
added at a molar ratio of 1:10, heme to nitrite. The reaction
mixture is heated at about 85.degree. C. (lower temperatures of
75.degree. and 80.degree. C. are also acceptable) for 15 min with
intermittent stirring, cooled in an ice bath to room temperature,
and centrifuged for 2 min at 3000 rpm. The supernatant is acidified
to pH 4 with 0.1M citric acid. Acidification can also be performed
before cooling and centrifugation. Use of acetic (0.1 M),
hydrochloric (0.1 M), phosphoric (0.2 M) or sulfuric (0.05 M) acids
as acidifying agents is also suitable. During acidification
solubilized proteins are precipitated. After centrifugation for 2
min at 3000 rpm (905.times. g), supernatant containing residual
nitrite from the process is discarded. Yield and purity of product
obtained from heat-treated cell/nitrite solutions are determined
after acidification of cooled reaction mixture to pH 4 and
extraction and recovery of the product from resulting precipitate
using 4:1 (v/v) acetone:water solution. Analysis of variance and
Student tests are used to determine differences in mean yield and
purity values based on data collected from three replications of
each treatment. Significance is determined at a 95% level of
probability.
Example 7
Enzyme and Other Means of Reduction
[0130] Another aspect of invention related to reduction of antigen
applies to agents specifically recognizing the amino acid
sequences.
[0131] To reduce antigen with the agents specifically recognizing
the amino acid sequences, a large number of site-specific agents
and their recognition sites are known and commercially available.
Examples are as follows (the cleavage sites are indicated with
".vertline." symbol): Hydroxylamine: Asn.vertline.Gly, formic acid:
Asp.vertline.Pro, acetic acid: Asp.vertline.Pro, BMPS-skatole:
Trp.vertline. and o-iodosobenzoic acid: Trp.vertline. are the
examples of chemical agents. Chymotrypsin: Trp.vertline. and
Phe.vertline., collagenase: Pro-X.vertline.Gly-Pro, endoproteinase
Lys-C: Lys.vertline., thrombin: Arg-Gly-Pro-Arg.vertline., and
trypsin: Arg.vertline. and Lys.vertline.are the examples of
proteolytic enzymes.
[0132] Bovine BSE antigen in the brain and bovine muscle tissue are
suspended in 0.1 M ammonium carbonate (concentration; 1 mg/ml),
heated for 30 min at 50.degree. C., and then digested with trypsin
(TPCK-Tripsin, Sigma Chemical Co., St. Louis, Mo., concentration,
2%) for 1 h at 37.degree. C. Enzymatic reaction is stopped by
adjusting pH to 3.0 with 12 N HCl and allowing to stand overnight
at 4.degree. C. Optionally undigested antigen and trypsin is
removed by ultrafiltration (Centriprep 3, Amicon, Beverly, Mass.).
Antigen digested with trypsin is also referred to as "reduced
antigen" in the description for a convenience' sake.
[0133] The tissue can be further reduced by sodium hypochlorite
(bleach) or by a detergent (SDS, phenol, polysorbate 80, Tween 20,
Tween 40, Tween 60, Zwittergent 3-12, Teepol HB7 or Span 85) or by
protein-crosslinking agent like formaldehyde. Other means of
reducing are known such as exposure to ultraviolet light or
irradiation by radioactive isotopes. Even exposure to sun or
ordinary indoor light for a sufficient period of time will reduce
the antigen and tissue into a desired state.
Example 8
Composition Obtained from Tissue or Cell Culture
[0134] It is also contemplated that instead of obtaining animal
tissues like blood or other tissue as the original source for
making the composition one skilled in the art can easily obtain an
equally effective composition by conventional tissue and cell
culture methods.
[0135] In one embodiment, the peripheral blood donated by healthy
volunteers is used as a source of human peripheral mononuclear
cells (PMNC). PMNC are isolated from blood using Histopaque (Sigma
Chemical Co., St. Louis, Mo.) density centrifugation at 1600 rpm
for 20 minutes. Isolated PMNC are washed three times with RPMI 1640
medium (J. R. Scientific, Woodland, Calif.) to remove residual
Histopaque. After the final wash, PMNC are resuspended in dye-free
RPMI 1640 containing 5% fetal bovine serum (Intergen, Purchase,
N.Y.). Cultures are incubated at 37 degrees Centigrade in 5%
CO.sub.2. About 1 to 7 days after inoculation with HIV, the
infected cells are harvested and processed including denaturing
steps to obtain a composition of the present invention.
[0136] Peripheral blood mononuclear cells obtained from
HIV-seropositive individuals are equally suitable. These cells can
be additionally immortalized by Epstein-Barr virus (EBV) infection.
Alternatively instead of PMNC or PBMC, immortalized cell lines can
be used for mass production of continuously replicating
HIV-infected cells. Such cells can comprise human cell lines H9,
MOLT-3, U937, HUT78, CEM, MT-2 among many others and are selected
from ATCC collection and propagated accordingly. One of skill in
the art can establish equally suitable, alternative cell lines cell
line, e.g., kidney cells of an African Green Monkey, by routine
art-known methods like inoculating said cell line with a sample
containing an inoculum of a virus; incubating said inoculated cell
line to permit proliferation of said virus; and harvesting the
virus. These and other cells and lines can be easily selected and
procured from public and private cell collections (see below).
[0137] In a similar manner when cancer vaccine is contemplated,
instead of fresh tumor cells obtained from a patient one can select
appropriate cancer cell line that derived from a cancer type
similar to type of tumor that patient is affected. One can select
just one cell line or use pooled non-identical cancer cell lines.
For example, to treat a patient suffering from a breast cancer one
can use either one or a plurality of pooled MCF-7, CAMA-1, SKBR-3,
or BT-20 breast tumor cell lines grown by a conventional method.
Literally thousands of such cell lines exist and these cell lines
are easily obtained from a large number of tumor cell sources,
e.g., American Type Culture Collection or ATCC (www.atcc.org) in
Manassas Va.; DCTDC Tumor Repository in Frederick, Md.; The
University of Michigan Breast Cell Line/Tissue Bank and Data Base
(http://www.cancer.med.umich.edu/umbnkdb.html); ECACC European
Collection of Cell Cultures (http://fuseiv.star.co.uk/camr); DSMZ
German Collection of Microorganism and Cell Cultures
(www.gbf-braunschweig.de/DSMZ); Fujisaki Cell Center or Japanese
Cancer Center (http://cellbank.nihs.go.j- p) both in Japan,
www.biotech.ist.unige.it/interlab/cldb.html in Italy; ECACC
European Collection of Cell Cultures in Salisbury, Wiltshire, UK
(www.camr.org.uk/frame.htm); The National Laboratory for the
Genetics of Israeli Populations (www.tau.ac.il/medicine/NLGIP);
Coriell Cell Depository (http://locus.umdnj.edu/nia) among many
others, the content of which is incorporated herein by way of
reference.
Example 9
Composition from Recombinantly Produced Pathogen
[0138] With advent of recombinant DNA technology it became possible
to employ procaryotic and eucaryotic hosts such as E. coli, yeast,
fungi, insect, and mammalian cells in culture to produce useful
antigens and fragments thereof. Gene expression is driven by
various promoters. Representative promoters suitable for use within
the present invention include both eukaryotic (e.g., pol I, II, or
III) and prokaryotic promoters, and inducible or non-inducible
(i.e., constitutive) promoters, such as, for example, Murine
Leukemia virus promoters (e.g., MoMLV), metallothionein promoters,
the glucocorticoid promoter, Drosophila actin SC distal promoter,
SV 40 promoter, heat shock protein 65 promoter, heat shock protein
70 promoter, immunoglobulin promoters, Mouse polyoma virus promoter
("Py"), rous sarcoma virus ("RSV"), BK virus and JC virus
promoters, MMTV promoter, alphavirus junction region, CMV promoter,
Adenovirus VA1RNA, rRNA promoter, tRNA methionine promoter, CaMV
35S promoter, nopaline synthetase promoter, and the lac promoter
among many others. Based on these one can easily imagine a gene
expression unit comprising a DNA coding sequence for a
non-bacterial heterologous protein selected from the group
consisting of a protein of mammalian origin and a protein of
mammalian virus origin, and a regulatory element for transcription
of said DNA sequence and translation within a Drosophila cell
wherein said regulatory element comprises a Drosophila
metallothionein promoter.
[0139] Transformed E. coli cells from a culture expressing high
levels (5%) of recombinant HIV antigens are streaked onto an
L-Broth plate containing 100 microg/ml ampicillin and the plate is
incubated overnight at 37.degree. C. A single colony is inoculated
into 10 ml of L-Broth, 100 micro/ml ampicillin and grown overnight
at 37.degree. C. An aliquot is used to verify plasmid structure by
restriction mapping with SalI and PstI. A second aliquot is used to
induce expression of HIV antigens and the rest of the culture is
made 15% glycerol by adding {fraction (1/4)} volume of 75% sterile
glycerol. Glycerol cell stocks are aliquoted in 1 ml and quickly
frozen in liquid nitrogen or dry-ice ethanol bath. These master
seed stocks are stored at -70.degree. C. When needed the master
seed stock is scraped with a sterile applicator which is used to
streak an L-Broth plate containing 100 microg/ml ampicillin. Single
colonies from this plate are used to inoculate 20-50 ml of
L-Broth/amp, which is incubated at 37.degree. C. overnight. An
aliquot of the overnight culture is used to inoculate larger
volumes (1-6 liters) of L-Broth/amp. Cells are incubated at
37.degree. C. overnight and reach an OD650 of approximately 5 prior
to use as inoculum for the fermenter run. Fermenters (capacity
about 16 liters) containing 10 l of L-Broth and 1 ml of antifoam
are inoculated with 100-500 ml from the inoculum culture. Cells are
grown at 37.degree. C. to an OD of about 1. Expression of HIV
antigens is induced by addition of 100 ml of an IPTG solution (100
mM) to yield a 1 mM final concentration in the fermenter. Cells are
grown for 3 additional hours and subsequently harvested using
continuous flow centrifugation. At this step cells can be frozen
and kept at -20.degree. C. until further proceedings.
Alternatively, 250 liter fermenters are inoculated with 1-5 liter
from the inoculum culture. Growth, induction, and harvest are as
indicated before. Frozen E. coli cells are thawed and suspended in
2.5 volumes of lysis buffer (0.1M sodium phosphate (NaPi), pH 7.5,
1 mM EDTA, 0.1 M NaCl). Cells are broken in a non-continuous system
using a 300 ml glass unit of a Dyno-mill at 3000 rpm and 140 ml of
acid-washed glass beads for 15 min. The jacketed chamber is kept
cool by a -20.degree. C. ethylene glycol solution. Broken cells are
centrifuged at 27,000.times. g for 25 minutes to remove debris and
glass beads. The supernatant is recovered and kept at 4.degree. C.
The cell extract is made 30% (NH4).sub.2 SO.sub.4 by slowly adding
the ammonium sulfate at 4.degree. C. The extract is stirred for 10
min after the final concentration is achieved, followed by
centrifugation at 27,000.times. g for 20 min. The pellet is
resuspended in 1 M NaCl, 1 mM EDTA, 1% Triton X-100, and 5% SDS,
and then boiled for 5 min. The fraction obtained by selective
precipitation is submitted to gel filtration using a G50 Sephadex
column equilibrated in 0.03M NaPi, pH 6.8. Chromatography is
developed in the same solution. Fractions are collected and
absorbance at 280 nm is determined. Antigen-containing fractions
are pooled and characterized by protein gel electrophoresis,
Western analysis, and ELISA and are further processed into the
composition of the invention. It is also equally possible to use
these recombinant antigens without these steps of purification.
[0140] Alternatively a process for purifying one or more antigenic
or immunogenic substances from a source liquid can comprise steps
of contacting the source liquid with a chromatography resin;
incubating the source liquid with the chromatography resin for a
sufficient contact time to allow the resin to bind a desired
fraction of one or more antigenic substances, recirculating the
chromatography resin in a cross-flow filter; concentrating the
chromatography resin and separating contaminants from the
chromatography-resin-bound antigens by concentration and/or
diafiltration; eluting the antigenic substance from the
chromatography resin; and separating the immunogenic substance from
the chromatography resin by diafiltration; recovering the desired
immunogen(s); and optionally concentrating the antigenic or
immunogenic substance.
Example 10
Usefulness of the Composition for Conducting Nonclinical
Studies
[0141] One of embodiments of the invention is the utility of this
composition for nonclinical studies. Those skilled in the art would
certainly be interested in studying as to how this composition
works. The mechanism of the action of the composition can be
studied in variety of ways in vitro and in vivo settings by routine
experimentation well known to those of skill in the art. Such
nonclinical studies can encompass toxicology, immunology, molecular
biology, pharmacology, metabolism, bioanalysis, pharmaceutical
analysis, and biosafety testing in support for further development
and improvement. One of ordinary skill in the art can easily find
relevant experimental protocols and general background information
on goals of these experiments by searching appropriate keywords on
many sites on Internet, e.g., http://www.1blink.com,
http://www.ihuge.com, www.pharmaceuticalonline.com,
http://www.mtdesk.com, www.wiley-vch.de/contents/ullmann,
wwwjimmunol.org, www.freemedicaljournals.com,
http://164.195.100.11/netah- tm1/search-bool.html,
http://www.ncbi.nlm.nih.gov/PubMed, the entire content of which is
incorporated herein by way of reference. Oral immune tolerance or
mucosal immune tolerance in wide sense of the term is a phenomenon,
which seldom causes immune responses against an antigen, if it is
taken orally. Since any antigen taken orally is absorbed through
the intestinal mucosa and then processed by functions of such
various tissues as Peyer's patch and epithelial cells of the
intestines and the adjacent lymphocytes, portal canal and liver,
administration of such antigen rarely causes allergic reaction or
shock. The oral immune tolerance has been examined as
immunosuppressive therapy for allergy and rejection in organ
transplantation. Intranasal administration of the antigen can also
induce immune tolerance, which causes no systemic immune response
to the antigen, since it is absorbed through the mucosa of the
digestive and airway tracts and the lungs.
[0142] For example such effects of immune modulation by instant
composition can be monitored by assessing cytokine production by
lymphocytes isolated from the mice intranasally given the antigen.
Cytokines, e.g., IFN-gamma and IL-10 related to inflammation, are
determined to examine the effects of stimulation with denatured
antigen on production of these cytokines. TGF-beta is known as
anti-inflammatory cytokine and immunologically important modulator.
DBA/1 mice are given intranasally under anesthesia 0, 0.002, 0.02,
0.2, 2, 20 or 200 microgram of denatured antigen derived from
tissues of Rous sarcoma virus infected mice which display visible
tumor mass due to virus infection. Alternatively these mice are
subcutaneously immunized in their hind paws with a mixture of
native antigen (100 microg) and Freund's complete adjuvant (Difco)
on day 0. Alternatively, DBA/1 mice are fed on a mixture consisting
of lyophilized and then powdered denatured antigen (4%) and
commercially available feed during a period of about 1 to 31 days
before testing the effect on immune system. After set number of
days, lymph nodes are aseptically excised from each mouse. Then,
cell suspensions of a single population are prepared and placed
(106 cells/well) in a 96-well microplate (Falcon). After adding
serum-free culture medium (X-vivo20, Biowhittaker) and antigen
(final concentration 500 microg/ml) to each well, the plates are
incubated for 3 days under 5% CO.sub.2. After that, the supernatant
fluid of the culture medium is collected. The control cells are
cultured under similar conditions without antigen or with
concanavalin A (final concentration 5 microg/ml) in place of
antigen. Since concanavalin A is a mitogen of T cells, it is used
to stimulate nonspecifically all the T cells as a positive control.
Rat monoclonal antibodies against mouse cytokines (anti-mouse IL-10
monoclonal antibody, JES5-2A5; anti-mouse IFN-gamma monoclonal
antibody, RA-6A2; Pharmingen) are coated (50 microl/well) on a
microplate (Maxisorp, Nunc). After washing with PBS-Tween, the
plate is blocked with 3% BSA-added PBS-Tween. Then, the
appropriately diluted culture supernatant described above are added
to each well. After washing with PBS-Tween, each of
biotin-conjugated rat monoclonal antibodies against mouse cytokines
(anti-mouse IL-10 monoclonal antibody, SXC-1; anti-mouse IFN-gamma
monoclonal antibody, XMG1.2; Pharmingen) is added to the wells.
After another washing with PBS-Tween, avidin-labeled alkaline
phosphatase (Zymed) is added. After washing with PBS-Tween, a
substrate solution (p-nitrophenyl-2-phosphate) is added to each
well and the mixture is allowed to stand for 60 min. The reaction
is stopped by adding 5 N NaOH. The concentrations of INF-gamma and
IL-10 produced are determined from the absorbance at 405 nm and
compared with those of the control samples. Other experimental
protocols in studying humoral and cell-mediated immune reactions
can be set up to study other aspects of composition and the
desirability of a particular protocol will depend on the nature of
investigation and particular aim of study of practitioners in the
art.
Example 11
Clinical Evaluation of the Composition in HIV-positive
Individuals
[0143] Thirty-eight HIV-infected individuals are given the instant
composition for several weeks (see results in Table 1). The
observed recovery is dramatic and beneficial results are observed
in a few days. Patients unable to get up from bed begin walking and
some actually return back to work. Remarkable weight gain is
observed, particularly with emaciated patients. A higher level of
energy and better mood is observed. The placebo effect is unlikely
because children are among the patients treated. Children do not
comprehend the meaning of the therapy. CD4 counts increase or
stabilize and CD8 counts--the markers associated with HIV-killing
cytotoxic T lymphocytes (CTL) rise steadily in almost every
patient. CTL activity is assessed in freshly isolated peripheral
blood mononuclear cells (PBMC) and in
phytohaemagglutinin-stimulated interleukin-2 expanded cell lines
established from PBMC. Cytotoxicity to HIV-1 env, gag, pol and nef
gene products is surveyed in a 4 h 51Cr-release assay using
autologous Epstein-Barr virus (EBV) transformed B cells infected
with vaccinia constructs expressing each of these HIV genes. The
immunodominant CTL epitope and MHC class I antigen restriction
specificity of HIV-specific CTL is mapped when present. Plasma
viral load is assessed by branched DNA assay or by RT-PCR. Attempts
are made to isolate virus from these individuals by the PBMC
coculture assay. This and other patient information comprising
gender, age, weight, CD4 information, viral load information, HIV
genotype and phenotype information, hemoglobin information,
neuropathy information, neutrophil information, pancreatitis,
hepatic function, renal function, drug allergy and intolerance are
collected by routine methods well known in the art.
[0144] Patients appear to be able to control their HIV infection by
showing vigorous cell-mediated immune response, evident from
increase in CD8 cell levels. The composition appears to provide an
unexpected benefit to the immune system and to the general well
being of the patients.
[0145] The dosage varies from patient to patient but commonly 1 to
5 tablets are taken orally every day. No anemia, usually associated
with antiviral treatment, is observed with treatment with this
composition. The following mild adverse reactions occur in about
50% of patients: mild allergic reaction eg. urticaria, chills,
fever, mild abdominal or back pain. No acute, severe allergic
reactions (anaphylactic or anaphylactoid) characterised by eg.
flushing of the skin, hypotension, substemal pain, bronchospasms,
dyspnoea, cardio-respiratory collapse are observed. Such mild
reactions can be easily controlled with antihistamines or
non-steroid or steroid inflammatory drugs. Usually, after 3-7 days
adverse reactions disappear without requiring medical
intervention.
[0146] Of interest is the observation that prostitutes who take
this composition only once or twice do not become infected during 6
months of observation, even though the rate of HIV infection among
this population is rather high. Statistically, this cannot be
attributed to coincidence.
3TABLE 3 Results of treatment of HIV patients with the composition
Tests No. Gender Age Hospital Weeks CD4 CD8 WBC Hemoglo Hemocrit Pt
1 F 23 CH 0 429 1168 7400 11.5 35.3 CH 19 670 2820 11200 12.3 36 Pt
2 M 27 CHU 0 12 392 5600 CH 19 20 520 7200 Pt 3 F 25 CHU 0 347 942
5900 13.1 40.1 CH 24 380 970 5300 13.7 40 ATRCS 54 Pt 4 M 28 CHU 0
32 294 3100 CH 7 10 460 4200 Pt 5 F 28 CHU 0 436 742 5240 CHU 27
647 1079 Pt 6 M 35 CH 0 0 300 5700 CH 8 0 690 4900 Pt 7 M 48 S 0
140 1390 CHU 8 174 1792 6600 Pt 8 M 36 S 0 108 1079 5700 CHU 29 241
1757 5900 Pt 9 F 35 S 0 440 520 CHU 18 551 735 4500 Pt 10 M 33 S 0
17 645 3300 CHU 29 57 614 4700 Pt 11 F 32 S 0 76 531 3700 CHU 29
101 495 4200 Pt 12 M 31 CH 0 530 1580 7800 15.1 43 CH 6 465 2030
8100 15.1 44 Pt 13 F 25 CH 0 230 530 5500 CH 5 320 690 6300 Pt 14 F
36 CH 0 230 530 4500 CH 9 210 1340 4300 Pt 15 F 41 R 0 270 900 5100
R 3 310 1070 6600 Pt 16 F 33 R 0 440 1680 6000 R 2 630 1720 5800 Pt
17 M 35 R 0 10 490 4600 R 2 10 520 3600 Pt 18 F 26 BPL 0 438 8500 V
34 360 1110 5200 ATRCS 45 Pt 19 M BA 0 141 BA 58 272 Pt 20 M 30 BPL
0 605 BPL 30 970 856 AI 32 9900 45 Pt 21 F 50 CH 0 400 1920 7600 CH
32 520 2330 8400 Pt 22 M 26 CH 0 0 240 3900 CH 9 0 350 Pt 23 F 31
CHU 0 240 888 6900 11.7 34.7 CHU 30 200 1077 7800 12 35.2 Pt 24 M
28 CH 0 0 180 5900 CH 38 0 100 22300 Pt 25 F CHU 0 218 629 9170
13.1 40.5 CHU 31 Pt 26 F 60 RA 0 175 437 RA 65 237 1163 5120 Pt 27
M 20 SR 0 195 5100 V 26 100 9600 Pt 28 F 35 CH 0 350 1560 9400 CH
28 280 1890 8000 ATCRS 44 Pt 29 M 37 YBON 0 30 1110 Pt 30 M Pt 31 M
34 CH 0 139 1034 Pt 32 F 30 CH 0 559 1737 Pt 33 M 4 CH Pt 34 M 27 V
0 160 7400 V 32 50 9100 Pt 35 F 39 Pt 36 M 32 CHU 0 195 1098 5400
14.9 45.9 CHU 24 140 1242 6000 14.8 44.5 Pt 37 F 34 CH 0 13 797
4600 6.9 23 ATRCS 8 Pt 38 F 56 CH 0 400 1920 7600 13.8 41 CH 23 540
2630 8800 14 42 HOSPITAL OR WHERE TEST IS CONDUCTED AI = Aikchol
ATRSC = Anonymous Treatment Red Cross Society B = Bangkok General
Hospital BA = Bangkok Lab BPL = Bangkok Pathology Laboratory CH =
Chonburi Hospital
Example 12
Shrimp Composition
[0147] Shrimp composition can be made by any of specific processes
disclosed supra or by using the protocol as follows. A species of
living shrimp are placed in fresh water or an aqueous salt solution
containing not greater than 0.3% by weight, preferably not greater
than 0.1% by weight, of at least one compound selected from organic
acids such as acetic acid, citric acid, succinic acid, malic acid,
tartaric acid and lactic acid; inorganic acids such as phosphoric
acid, sulfuric acid and hydrochloric acid; and sodium or potassium
salts of these acids. The aforesaid solution can also be
characterized as a slightly acidic aqueous solution having a pH of
3 to 6.5. These living shrimp are left therein at a temperature of
1.degree. to 25.degree. C., preferably 2.degree. to 15.degree. C.,
for a period of 0.5 to 72 hours, preferably 1 to 40 hours. Thus,
the alimentary canal of the living shrimp is substantially freed of
soil by their own excretory power. Then, the living shrimp are
washed with water to remove any dirt from the body surfaces
thereof. These washing steps are however optional. Thereafter, the
living shrimp are wet-ground and the resulting suspension is frozen
at a low temperature of -5.degree. C. or below, preferably
-10.degree. to -60.degree. C. Then, the frozen suspension is
freeze-dried and vacuum-dried. Specifically, while the temperature
is raised stepwise from -60.degree. to +90.degree. C., preferably
from -40.degree. to +80.degree. C., the suspension is freeze-dried
and then vacuum-dried under a vacuum of 100 mmHg or below,
preferably 30 mmHg or below, for a period of 5 to 100 hours,
preferably 10 to 60 hours. Thus, there is obtained sterile dried
shrimp powder. To insure the denaturation the powder is then
autoclaved for about 20 minutes at 121.degree. C.
[0148] In the step of wet-grinding the shrimp, i.e., the step of
destroying the tissues (or cells) of the shrimp, it is preferable
to form the shrimp into a suspension or homogenate by means of a
suitable device such as homogenizer, blender, homomixer, smasher or
pressurized cell destroyer. This wet-grinding step is desirably
carried out at a temperature of 1.degree. to 25.degree. C. and
preferably 2.degree. to 15.degree. C. Alternatively, shrimp is
dried first by freeze-drying and then ground into fine powder.
[0149] According to either of the above-described processes, dried
shrimp powder having a yellowish or brown color can be obtained
from shrimp. In ordinary cases, the dried shrimp powder is prepared
so as to have a moisture content of 5 to 16%, preferably 7 to 14%,
an ash content of 3 to 8%, preferably 4 to 7%, and a nitrogen
content of 1 to 11%, preferably 6 to 11%. The dried shrimp powder
thus obtained contains baculovirus antigens and shrimp tissues.
[0150] The composition is equally suitable for preventing the
mortality due to disease in commercially farmed fish like salmon or
trout, e.g., Fish Pancreas Disease Virus (FPDV). This industry
involves not thousands of dollars, but hundreds of millions of
dollars. The present invention provides a method for the
protection, service to and salvage of a multibillion dollar
industry; and also aids in providing a vaccine to a world where
maintenance of the food supply is becoming an ever increasing
problem.
Example 13
Use of the Composition as a Preventive Vaccine
[0151] Shrimp is a crustacean which belongs to the Genus Penaeus.
Wild shrimp has been caught for food for centuries. Many species of
shrimp can be nowadays cultivated, e.g., highly praised Tiger Prawn
(Penaeus monodon, Fabricius). Shrimp farming is a worldwide
industry with a large market and approximately half of the shrimp
consumed in the United States is from farms. While shrimp farming
is a lucrative business, there are serious drawbacks associated
with farming the dense populations of shrimp in close quarters, of
particular concern is the susceptibility of these animals to viral
and bacterial infections.
[0152] One of many viral infections is caused by Monodon
baculovirus (MBV) which belongs to Baculoviridae family of viruses
which preferentially replicate in insect tissues. The infected
prawn display yellow cream colored hepatopancreas and expand in
size. The exterior appearance is also yellow in color and thus the
Baculovirus-caused disease is sometimes referred to as Yellow Head
Disease. Other pathogenic Baculoviruses are also known such as
Baculovirus penaei (BP), Baculovirus mid gut gland necrosis (BMN).
These viruses infect diverse species of prawn such as P. japonicus,
P. duorarum, P. stylirostris, P. vannamei, P. aztecus, and P.
marginatus among many others. Moreover the Hepatopancreatic
parvo-like virus (HPV) infects P. merquiensis species in Australia,
P. orientalis in Singapore, P. chinensis in China and P. monodon.
in Philippines. In addition to viruses pathogenic microbial
infections in shrimp are also common, especially those caused by
family of Vibrio and Aeromonas, e.g., vibrio diseases of Kuruma
prawn (Penaeus japonicus), Giant tiger prawn (Penaeus monodon),
White-leg shrimp, Vibrio anguillarum, or the like; parasitic
diseases of Epistylis sp., Soothamnium sp., Ichthyopthiriasis or
the like; mycotic infectious diseases of Lagenidium sp., Siropidium
sp., or the like. The present composition prepared according to the
art-known process selected from one of disclosed examples and
comprises denatured (non-live) Baculovirus-infected shrimp tissues
and Baculovirus antigens.
[0153] The obtained composition appears as a freely flowing powder
and individual particles exhibit a crystalline appearance. This
preparation is then spiked into a standard shrimp meal at a desired
concentration. Typical shrimp meal consists of Fish head meal,
Squid meal, Soybean meal, Cereal products or by-products, Fish oil,
Soybean lecithin, Cholesterol, Binder, Dicalcium phosphate,
Vitamins, and Trace minerals mix. Usually, the effective dose of
composition ranges from about 0.01 to 500 gram per kg of shrimp
meal, preferably from about 0.1 to 20 gram, more preferably from
about 6 to 16 gram, but the final and optimal dose may vary
depending on desired result and aquaculture conditions.
[0154] Shrimp meal prepared by this process is fed either to
non-infected shrimp population in the aquaculture or to already
infected sick shrimp. The composition is equally effective in both
circumstances.
[0155] FIG. 1 provides results based on use of composition as a
preventive or prophylactic preparation or vaccine. Freshly seeded
shrimp larvae that have not been fed with the composition contract
the virus present in the water and die as a result by the end of 8
weeks. By contrast as can be seen from FIG. 1 there is a clear
dose-dependent survival rate among shrimp fed with increasing
concentration of the composition.
[0156] About 1 kg of shrimp meal is adequate per about 100,000
shrimp aged from about 1 to 15 days and meal particle size for this
age is about 100 micron. Correspondingly, it is preferable that
about 1.3 kg will be sufficient per 100,000 shrimp aged 20 days and
meal particle size at this stage can be slightly bigger at about
200 micron. Similar adjustments can be made depending on age and
population size of shrimp, e.g., about 1.8 kg/100,000 age 25 days
at meal size about 200 micron; 2.5 kg/100,000 age 30 days at meal
size 200 micron; 3.5 kg/100,000 age 40 days at meal size 300
micron; 4.0 kg/100,000 age 50 days at meal size 300 micron; 5.0
kg/100,000 age 60 days at meal size 400 micron, and alike. The
composition is administered about 8 times during the period of
vaccination, although the frequency of feeding and meal quantity
can vary depending on desired result.
[0157] Thus while various alterations in the protocol can be made
and established by routine experimentation familiar to those of
skill in the art, this example illustrates clearly the broad
applicability of the inventive concept across a wide variety of
animal species.
Example 14
Microbial Infection of Unknown Etiology
[0158] Every year new pathogenic microbes and viruses are added to
the growing list of infectious diseases. Since 1973 many new
species have been added, e.g., 1973 Rotavirus Virus Major cause of
infantile diarrhea; 1975 Parvovirus B19 Virus Aplastic crisis in
chronic hemolytic anemia; 1976 Cryptosporidium parvum Parasite
Acute and chronic diarrhea; 1977 Ebola Virus Ebola hemorrhagic
fever; 1977 Legionella pneumophila Bacteria Legionnaires' disease;
1977 Hantaanvirus Virus Hemorragic fever with renal syndrome
(HRFS); 1977 Campylobacter jejuni Bacteria Enteric pathogens
distributed globally; 1980 Human T-lymphotropic Virus T-cell
lymphoma-leukemia virus I (HTLV-1); 1981 Toxic producing strains of
Staphylococcus aureus Bacteria Toxic shock syndrome (tampon use);
1982 Escherichia coli 0157:H7 Bacteria Hemorrhagic colitis;
hemolytic uremic syndrome; 1982 HTLV-II Virus Hairy cell leukemia;
1982 Borrelia burgdorferi Bacteria Lyme disease; 1983 Human
immunodeficiency virus (HIV) Virus Acquired immunodeficiency
syndrome (AIDS); 1983 Helicobacter pylori Bacteria Peptic ulcer
disease; 1985 Enterocytozoon bieneusi Parasite Persistent diarrhea;
1986 Cyclospora cayatanensis Parasite Persistent diarrhea; 1988
Human herpesvirus-6 (HHV-6) Virus Roseola subitum; 1988 Hepatitis E
Virus Enterically transmitted non-A, non-B hepatitis; 1989
Ehrlichia chafeensis Bacteria; Human ehrlichiosis; 1989 Hepatitis C
Virus Parenterally transmitted non-A, non-B, liver infection; 1991
Guanarito virus Virus Venezuelan hemorrhagic fever; 1991
Encephalitozzon hellem Parasite Conjunctivitis, disseminated
disease; 1991 New species of Babesia Parasite Atypical babesiosis;
1992 Vibrio cholerae 0139 Bacteria New strain associated with
epidemic cholera; 1992 Bartonella henselae Bacteria Cat-saatch
disease; bacillaryangiomatosis; 1993 Sin nombre virus Virus Adult
respiratory distress syndrome; 1993 Encephalitozoon cuniculi
Parasite Disseminated disease; 1994 Sabia virus Virus Brazilian
hemorrhagic fever; and in 1995 HHV-8 Virus Associated with Kaposi
sarcoma in AIDS patients among many others.
[0159] The present invention is particularly useful in treating and
preventing viral and bacterial infections in which the causative
pathogen has not been identified and thus no therapy is available.
This composition is equally suitable for treating pathogens of
known origin but for which no therapy has yet been developed.
[0160] For instance, a colony of captivated rhesus monkeys in a zoo
begin dying from a rapidly evolving disease. The symptoms of
jaundice and haemorrhagic manifestations are observed (epistaxis,
haematemesis, melaena and uterine bleeding) followed by
albuminuria, coma and death two to three days later. An infection
with unknown virus is suspected and normally the only available
remedy in this situation is the quarantine and destruction of the
entire colony. However, one monkey is bled and a composition is
prepared from the blood of the infected animal. This composition is
then fed to five sick monkeys who rapidly recover. The composition
is administered to all sick as well as non-infected monkeys.
[0161] A larger batch is made from pooled blood of several infected
primates and this preparation is administered orally to the entire
colony.
Example 15
Influenza Vaccine Preparation
[0162] Fertilized chicken eggs are inoculated with a strain of
Influenza prevalent during current season, e.g., virus Type B or
subtypes H1N1 and H3N2 of Influenza Type A. The inoculation is
carried out by art-established procedures, i.e., by injection of a
small volume of virus into the allantoic cavity. In the prior art
very extensive purification steps are required to separate the
virus from egg protein. According to the principles of this
invention this step is not anymore required. The whole mass of egg
liquid is obtained by breaking the shells of eggs and is simply
spray-dried at a blowing temperature of 175.degree. C. This
reduction step results in dry egg powder comprised substantially of
spherical particles having an average particle diameter of 100
micrometer. The powder is then optionally autoclaved or in the
absence of an autoclave the dry egg powder is spread on a cloth
laid over a steamer and exposed to steam at 100.degree. C. for 2
minutes to conduct heat denaturation, thereby to produce the
processed egg powder of the present invention. At the upper part of
the steamer is provided a cover for preventing dropping of
condensed water on the egg powder. Alternatively the processed egg
powder is added quickly to an equivalent weight of boiling with
stirring in a mixer, and the mixture is left standing for about
3-10 minutes. These products are then prepared as conventional egg
powder product and given to as a breakfast supplement to vaccinate
humans against seasonal influenza outbreak.
[0163] This composition can be equally applied for veterinary
purposes, e.g., for treating or preventing RNA-containing equine
influenza viruses, which belong to the family Orthomyxoviridae.
Equine influenza virus, and other unrelated pathogens like equine
protozoan myeloencephalitis or whooping cough.
Example 16
Malaria Vaccine
[0164] Malaria is one of the world's biggest killers--more than 2
million people die from it every year, and hundreds of millions are
infected. While most infectious diseases are caused by bacteria or
by viruses, malaria is caused by a Plasmodium endoparasite, which
enters into host's body with mosquito bites. Despite intensive
research a vaccine is yet to be produced. Several vaccines have
been developed based on the Circumsporozoite (CS) protein but all
of them failed since they are focused on one or few antigens.
[0165] If malaria infected mosquito is prepared as a composition
according to principles of the invention and given to naive
subjects they can develop immunity against malaria.
[0166] The present composition prepared according to instant
principles comprises over 2000 proteins and even higher number of
antigens synthesized by asexual stage malaria in mosquito gut
tissues. Subjects taking the instant preparation orally fail to
become infected with malaria. Infected patients who are given the
ground mosquito pills show signs of recovery from the disease.
Hence, the present composition is effective as a therapeutic and a
preventive remedy not only against viruses and bacteria but also
against malarial parasites, especially those spread by Anopheles
sp., i.e., Plasmodium falciparum, P. malariae, P. vivax, and P.
ovale.
[0167] In considering these results it is apparent that other
arthropod-borne pathogenic diseases either viral, bacterial, or
protozoan or other parasites can be treated. These include but are
not limited to those spread by Mite: Leptotrombidium sp. (red
mites) Scrub Typhus (Tsutsugamushi disease) Rickettsia
tsutsugamushi (bacteria, intracellular); Mite: Liponyssiodes
sanguineus (mouse mite) Rickettsial pox Rickettsia akari
(bacteria); Tick: Dermacentor sp. Tularemia Francisella tularensis
(Gram negative bacteria); Tick: Dermacentor sp. and other Ixodid
ticks Rocky Mountain Spotted Fever Rickettsia rickettsia
(bacteria); Tick: Ornithodoros sp. Endemic Relapsing Fever Borrelia
sp. (bacteria, spiral shaped); Tick: Ixodes sp. Babesiosis Babesia
microti (parasite, protozoan); Tick: Ixodes sp. Lyme disease
Borrelia burgdorferi (bacteria, spiral shape); Tick: Dermacentor
variabilis, Amyblyomma americanum Ehrlichiosis, Sennetsu fever
Ehrlichia canis E. sennetsu E. chaffeensis E. equi E.
phagocytophilia (bacteria, intracellular); Tick: Dermacentor sp.
Colorado Tick Fever CTF virus, Eyach virus, or strain S6-14-03
(Reoviridae); Tick: Russian Spring-Summer Encephalitis, Louping Ill
Encephalitis, Langat Encephalitis, Powassan Encephalitis, Omsk
hemorrhagic fever Russian Spring-Summer Encephalitis, Louping Ill
Encephalitis, Langat virus, Powassan virus, Omsk hemorrhagic fever
virus (Flaviviridae); Tick: Nairobi Sheep fever, Crimean
hemorrhagic fever Nairobi sheep disease virus, Crimean-Congo
hemorrhagic fever virus (Bunyaviridae); Crustacea Copepod: Cyclops
sp. Diphyllobothriasis, fish tapeworm Diphyllobothrium latum
(parasite, cestode, tapeworm); Strongyles, Ascarids, hookworms,
lungworms, filarial worms, whipworms; Copepod: Cyclops sp.
Sparganosis Diphyllobothrium spirometra (parasite, cestode,
tapeworm); Copepod: Cyclops sp. Dracunculosis Dracunculus
medinensis; Crabs, crayfish: various freshwater species
Paragonimiasis Paragonimus westermani; Lice: Pediculus humanus
Epidemic typhus Rickettsia prowazekii (bacteria); Lice: Pediculus
humanus Trench fever, bacillary angiomatosis, bacillary peliosis
Bartonella quintana (Gram negative bacteria); Lice: Pediculus
humanus Louse-borne relapsing fever or epidemic relapsing fever
Borrelia recurrentis (bacteria; spiral shape); Flea: Xenopsylla
cheopis, and various other rodent fleas Plague Yersina pestis (Gram
negative rod shaped bacteria); Flea: Xenopsylla cheopis Murine
typhus Rickettsia typhi (bacteria); Flea: Xenopsylla cheopis, and
various other rodent fleas Rat tapeworm infection Hymenolepsis
diminuta (parasite; cestode; tapeworm); Flea: various species Dog
tapeworm infection, Dipylidiasis Diphylidium caninum (parasite;
cestode; tapeworm); Bug: Triatoma species, Panstrongylus sps
(Kissing assassin bug, Reduvid bug) Chaga's disease Trypanosoma
cruzi (parasite; protozoan); Beetles: flour beetle Hymenolepsis
Hymenolepsis nana (parasite; tapeworm; cestode); Fly, gnat:
Glossina sp. (tsetse fly) African trypanosomiasis, African sleeping
sickness Trypanosoma brucei rhodesiense and T.b. gambiense; Fly,
gnat: Simulium sp. (black fly) Onchocerciasis, River blindness
Onchocerca volvulus (parasite; round worm; nematode); Fly, gnat:
Chrysops sp. Tularemia Francisella tularensis (Gram negative rod
shaped bacteria); Fly, gnat: Phlebotomus sp., Lutzomyia sp.
(sandflies) Leishmaniasis Leishmania donovani (Visceral, dumdum
fever, kala-azar), L. tropica (cutaneous; oriental sore, Delphi
boil), L. braziliensis (mucocutaneous; espundia, american
leishmaniasis, chiclero ulcer) (parasite; protozoan); Fly, gnat:
Phlebotomus sp. (sandfly in Peru, Ecuador and Columbia)
Bartonellosis, Oroya fever, Carrion's disease Bartonella
bacilliformis (Gram negative bacteria); Fly, gnat: Chrysops sp.
(mango flies) Loaiasis, Eye worm Loa 10a (parasite; nematode;
roundworm); Fly, gnat: sandfly Sandfly fever, Rift Valley fever
Sandfly fever Naples virus, Sandfly fever Sicilian virus, Rift
valley fever virus (Bunyaviridae); Mosquito: various species
Bancroftian filariasis, filarial Elephantiasis Wuchereria bancrofti
(parasite; nematode; roundworm); Mosquito: various species Malayan
filariasis, filarial Elephantiasis Brugia malayi (parasite;
nematode; roundworm); Mosquito: various species Dirofilariasis
Dirofilaria immitis (parasite; nematode; roundworm); Mosquito:
Aedes aegypti Yellow fever Yellow fever virus (Flaviviridae);
Mosquito: Aedes sp. Dengue fever, Break Bone fever Dengue fever
virus (Flaviviridae); Mosquito: Culiseta melanura, Coquillettidia
pertubans, Aedes vexans Eastern Equine encephalitis Eastern Equine
Encephalitis virus (Togaviridae); Mosquito: Aedes triseriatus La
Crosse encephalitis La Crosse Encephalitis virus (Bunyaviridae);
Mosquito: Culex sp. St. Louis encephalitis St. Louis Encephalitis
virus (Flaviviridae); Mosquito: Culex sp., Culex tarsalis
Venezualan equine encephalitis, Western equine encephalitis
Venezualan Equine Encephalitis virus, Western Equine Enchephalitis
virus (Togaviridae); Mosquito Chikungunya forest fever Chikungunya
virus, Mayaro fever, Mucambo fever, O'Nyong-Nyong fever, Pixuna
fever, Ross River fever (Togaviridae) Mosquito fevers and
encephalitis Nile fever, Japanese encephalitis virus, West Nile
fever, Zika fever, Wesselsbron fever, Kyasanur forest disease virus
(Flaviviridae); Mosquito fevers and encephalitis Oropouche virus,
Bunyamwera, Bwamba fever, Guama fever, Oropouche fever, California
Enchephalitis virus (Bunyaviridae); Mosquito fevers Chandipura
fever, Piry fever (Rhabdoviridae) among many others.
Example 17
Fibromyalgia
[0168] The subject a 27 years old female is presented with
fibromyalgia. Symptoms included chronic fatigue, stomach disorders,
pain and swelling in all joints, amenorrhea and swelling in the
breasts. The patient is diagnosed approximately five years before
beginning treatment. Patient had been taking on a daily basis 650
mg of tylenol and formulation of tylenol with codeine. The
subject's 24-hour base pre-therapy prolactin levels are high
throughout the day. For the first 4 weeks of treatment, the patient
is administered 625 mg of composition which is prepared from small
sample of joint biopsy according to the process of the invention.
Prolactin profiles are measured and show that the patient's daytime
prolactin levels is reduced. The clinical improvements in this
patient also includes discontinuation of both tylenol and codeine,
and reduction in the following symptoms: fatigue, stomach disorders
and pain in all joints. In addition, a normal menstrual cycle is
restored and swelling of breasts significantly subsided.
Example 18
Crohn's Disease
[0169] 20 years old male is presented who is diagnosed with Crohn's
disease based on exploratory surgery and barium X-ray.
Approximately 12 inches of the small intestine show inflammation.
The subject initially received prednisone 40 mg/day which did not
made any improvement to his condition. A composition is then
prepared using tissue obtained from surgery. After two months of
daily intake of one pill prepared of intestinal tissue the clinical
improvements to this patient include following: (1) avoidance for
surgical resection within this time period; (2) no increase in
inflamed area of intestine despite discontinuance of prednisone,
based on a comparison of X-rays from first diagnosis with most
recent; (3) during the time from first diagnosis to end of
treatment scarring is minimal as determined by intestinal response
to prednisone treatment; and (4) the patient reports no major
intestinal discomfort during treatment despite no major dietary
changes from pre-diagnosis.
Example 19
Prevention of Kidney Transplant Rejection
[0170] In this experiment a right nephrectomy is performed on 10
mice at the same time an allograft (kidney transplant from a
different strain of mouse) is performed. All ten of the animals are
observed for the first five days without immunosuppressive therapy.
Non-treated control animals receiving a nephrectomy and an
allograft kidney transplantation, exhibit initial signs of severe
rejection around day 5 post-transplantation. Five of the animals
(treated group) are given daily along with standard food pellets a
small dose of formulated (denatured and crystallized powder of
murine kidneys admixed and made into food pellets) composition for
the next five days. Animals are fed ad libitum but the consumed
dose does not usually exceed 10 mg of pure kidney composition per
day. All five treated animals experience rapid reversal of their
rejection symptoms, including a return to normal levels of
creatinine and eventually live beyond 100 days. The untreated
control animals are all dead by day ten due to acute organ
rejection. These results with respect to avility to overcome the
immune rejection are significant in confirming that the present
composition is highly effective in suppressing an inflammatory
immune response.
Example 20
Anti-STD and Contraceptive Product
[0171] The present invention can be useful against pathogens for
sexually transmitted diseases (STDs) of viral and bacterial origin
as well as a new prophylactic contraceptive method. For women,
intrauterine devices (IUDs) and intravaginal devices (IVDs) can be
designed that release antigens into the uterus or into the vagina
to provide continuous priming/protection against pregnancy and STDs
for periods of months to years. For example for men a skin lotion
can be formulated containing sperm and STD antigens to be applied
to the penis and external genitals during sexual activity, thereby
delivering prophylactically effective doses of antigen to virtually
all areas of skin and epithelia across which most STDs, including
AIDS, are usually transmitted, and, during vaginal or rectal
intercourse, the penis will deliver a contraceptively reliable dose
of the sperm immunogen to the cervical region of the vagina.
Example 21
Kaposi Sarcoma (KS) Treatment
[0172] Kaposi's sarcoma, the most common malignancy in AIDS
patients. HIV-seropositive patients with confirmed KS that
progressed over several months before enrollment are given an
initial dose of 850 mg/day of oral composition of the invention in
an open label study for 2 months. Toxicity, tumor response,
immunologic and angiogenic factors, and virologic parameters are
assessed on a regular basis. Twenty patients aged 21 to 47 years
with a median CD4 count of 240 cells/mm.sup.3 are enrolled. All
patients are assessable for toxicity and response. Minor stomach
and intestinal disturbances (diarrhea) in seven patients and mild
and transient headache in three patients are the most frequent
notable symptoms of toxicity. Positive side-effects include
improved mood, libido, and lean body mass weight gain. Eight (47%;
95% confidence interval [CI], 23% to 72%) of the 17 assessable
patients show a complete response, and additional two patients a
partial response consisting of no-progression in size or number of
lesions and drying of moist lesions. Based on all 20 patients
treated, the response rate is over 50% (95% CI, 19% to 64%). The
median composition dose at the time of response is 850 mg/day. The
median duration of drug treatment is 6 months. Thus, oral
composition of the invention is well tolerated in this population
of patients for as long as 24 months and is found to induce
clinically meaningful anti-KS responses in a sizable subset of the
patients. It is of particular importance that instant composition
useful in treating KS is the same composition useful in treating
AIDS patients in Example 11.
[0173] Thus composition and method for cancer therapy useful in
treating human patients with tumors to inhibit recurrence and
formation of metastases. This will for example comprise surgically
removing tumor tissue from a human cancer patient, reducing the
tumor tissue to small fragments, e.g., powder, denaturing the
fragments, formulating into a pill, and administering the vaccine
orally into the human patient for a period of time sufficient,
e.g., 5 years, to assure that metastases or cancer does not
recur.
Example 22
Tuberculosis Treatment
[0174] Mycobacterium tuberculosis or Tuberculum bacilli (T. B.)
grows in the endobronchial space and is found in the sputum of
infected individuals. During exacerbations of infection, such
growth also occurs in the alveoli. Tuberculosis is a highly
infectious disease that is characterized by the inflammation and
progressive destruction of lung tissue. The debilitation of the
lungs in patients with tuberculosis is associated with accumulation
of purulent sputum produced as a result of chronic endobronchial
infections caused by Mycobacterium tuberculosis. Nearly all
individuals suffering from tuberculosis eventually die of
respiratory failure. Presently, administration of drugs, such as
aminoglycosides kanamycin, streptomycin and amicacin as well as
isoniazid is the treatment of choice for tuberculosis patients.
However, penetration of these drugs into the bronchial secretions
is poor and treatment is often ineffective especially against drug
resistant bacteria. Sputum from tuberculosis patients is collected
and prepared into anti-tuberculosis therapeutic vaccine in a manner
similar to vaccine preparation from a blood. Aerosolized substance
is then administered with aid of jet nebulizers, e.g., Sidestream
(Pari Respiratory Equipment, Richmond, Va.). Patients receive
serial doses of 300 mg of the composition (5 mL of a 60 mg/mL
solution) from jet nebulizers. Twenty patients are enrolled in each
study. Each patient receives, in random order, placebo or active
administration from nebulizer delivery system. The doses are
separated by at least 2 days and not more than 5 days. Airway
irritation and acute bronchospasm are assessed by measuring
spirometry immediately prior to and 30 min post-completion of
aerosol administration. A decrease in forced expiratory volume
>15% in the 30 min spirometry test is considered evidence of
bronchospasm.
[0175] All patients with underlying disease of tuberculosis,
confirmed at entry by the inclusion/exclusion criteria specified in
this protocol, are eligible for enrollment into the study.
Investigators at the participating TBC centers select patients that
meet all of the inclusion criteria and one of the exclusion
criteria. Eligible patients are admitted to the study center on the
day of the study and receive aerosol therapy if they fulfill
entrance criteria. Physical exam is administered by a physician or
RC nurse prior to initial aerosol treatment only. Vital signs,
height, weight, oximetry, assessment of current respiratory status
and brief medical history are recorded. Patients sat upright and
used nose clips during the aerosol administration. The total
duration of time and the number of inhalations required to complete
the aerosol treatment are recorded. Any evidence of wheezing or
respiratory distress are recorded as well as number of rest periods
required by the subject because of dyspnea or excessive coughing
during the administration period. Following the last aerosol
treatment of the study, patients receive a physical exam to
evaluate clinical improvement.
[0176] One does not need to follow exactly the procedure described
above and can instead of collecting a sputum or bronchial lavage
order a necessary microorganism from a depository. Numerous
depositories exist wherein one can easily select desired pathogen,
e.g., The Belgian Coordinated Collections of Microorganisms (BCCM)
at www.belspo.be/bccm; The Culture Collection of Algae and Protozoa
at www.ife.ac.uk/ccap; Dutch Centraalbureau voor schimmelcultures
at www.cbs.knaw.nl; Microbial Information Network of China at
www.im.ac.cn among many others, the content of which is
incorporated herein by way of reference.
Example 23
Pyoderma Treatment
[0177] Patients with pyoderma, including furunculitis, cellulitis,
and folliculitis, are treated with the composition along with a
control group which is not treated. Medications are administered
either orally or intranasally for 5 consecutive days. Immunological
indices are normalized with disappearance of skin manifestations
and relapses are prevented after treatment with instant. Clinical
improvement correlate with immunological indices correction.
Administration intranasally or topically as a sterile saline
solution of medication for a period of 5 to 10 days at a
concentration of 1 mg/kg body weight. A number of patients within
the group patients afflicted with furunculitis, pyoderma,
cellulitis, and folliculitis are afflicted with acne vulgaris and
acne. The immunological indices are corrected and normalized
rapidly within the group therapy. The clinical outcome correlates
with the correction of immunological indices, and relapses are
controlled.
Example 24
Pelvic Inflammation
[0178] Female patients with the various disorders (pelvic
inflammatory diseases, cervicitis, vaginitis and various
tubo-ovarian and adnexal abscesses) are treated and some patients
are used as controls. The composition is given intravaginally at
100 mg for 5 consecutive days or 50 mg intranasally for 5
consecutive days in conjunction with conventional therapy. The
clinical effect of is expressed as the arresting of pain syndrome,
the control of body temperature, e.g. reduction of fever, the
decrease of duration of conventional treatment. The normalization
of immune status correlates with clinical improvements.
Example 25
Herpes Outbreak
[0179] Patients treated with the composition topically or
intranasally. They experience marked reduction of recurrence of
herpetic lesions, with substantial reduction in the period between
outbreaks. Treatment in combination with interferon also shows a
lessening of lesion outbreaks.
[0180] In a separate trial patients with Herpes Zoster are treated
with the composition in combination with conventional interferon
treatment and some control patients with interferon alone.
Administration single daily oral or intranasal 100 mg dose during a
period of 10 days results in accelerated regression of foci of
herpetic infection. The healing occurs earlier than in control
groups.
Example 26
Gingival Disease
[0181] Patients are treated for gingival disease by buccal
administration of the composition in the area of the gingiva. The
treatment results in the arresting of gingival disease.
Administration of 100 mg bucally or by electrophoresis (whereby a
small voltage charge to the gums results in a rapid transfer of
medication through the gum epithelium) results in the arresting of
bleeding, more rapid restoration of inflammatory processes, and the
decrease of purulent discharge. The treatment results in fewer
recurrences of the disease and appearance of normal gums for longer
time period than in controls.
[0182] The treatment with toothpaste containing the composition
also results in a reduction of dental caries.
Example 27
Acute Respiratory Disease
[0183] Patients with acute respiratory disease, including upper
airway diseases, such as colds, are treated with the composition.
Administration per os, intranasally or and installation into
sinuses of 100 mg doses for 3-7 days results in a milder course of
the viral infection. Secondary infectious complications are
diminished, and the duration of the treatment is also diminished.
Normalization of nasal breathing, the disappearance of nasal mucous
swelling, the arresting of exudates from affect sinuses, and
improved general condition and immune status is a common
observation. Accelerated reduction in symptom complexes including
joint pain, muscle aches, fevers, chills, and upper respiratory
symptoms is also common.
Example 28
Ocular Diseases
[0184] Patients with various eye problems are treated by
conventional methods, with one group receiving the composition in
addition to the conventional treatment. Administration of the
composition intraocularly at 18 mg for 5 consecutive days, or as
installation into conjunctival cavity as drops for 5 days results
in more rapid arresting of the inflammatory process and the
increase in visual acuity, and the decrease of duration of
treatment.
Example 29
Treatment of Wounds
[0185] The composition is used in patients with wounds of various
origin, type and localization. The compound consisting of wound
biopsy is administered topically as a single dose daily at 100 mg
for 10 days. The use of the formulation speeds up (when compared to
the control group) significantly wound healing, reduces therapy
duration and prevents the development of infectious complications.
Statistically fewer infections and reduced scarring is
observed.
Example 30
Flu Vaccine Co-administration
[0186] The product is given to persons in combination with the
anti-flu vaccination delivered by air pressure. The compound dose
is 50 mg delivered in a single oral dose for 3 consecutive days.
After product use, a significant decrease of sickness rate for a
period of 12 months is observed compared to controls who receive
flu-vaccination alone without the composition.
Example 31
Shigella
[0187] A total of 10 patients infected with Shigella dysentery are
examined. The composition prepared from stool sample of one the
patients is administered orally in doses of 100 mg for 10
consecutive days with resultant normalization of fever, the
reduction of toxemia, and the normalization gastrointestinal
disorders and symptoms.
Example 32
Oral Rabies Vaccine for Dogs
[0188] The freeze-dried composition consisting of commercial
vaccine suspension of SAG-2 strain of rabies virus of appropriate
titer along with host cells in which virus is propagated is added
at about 10 g per 100 g of dry mixture which has the following
ingredients: lactose (28%); mannitol (70%); gum arabic (2%). The
preparation is then dipped in a homogeneous mixture maintained at
56-60.degree. C. of the following composition: paraffin 50.degree.
C. (52%), ethylenevinyl acetate copolymer (28%), vinyl acetate
(6%), meat meal (23%), beef fat (17%), ROBERTET bacon flavor (2%).
A dosage form is thus formulated into a product that has an
appearance of an ordinary dog food pellet and is suitable for oral
vaccination against rabies according to the invention.
Example 33
Dental Composition
[0189] A premix containing the active ingredients and excipients
and having the following excipient composition: chlorhexidine
diacetate (0.25%), methionine (3%), lactose (80%),
beta-cyclodextrin (13%), dextran 70 (2%), pulverized silica
(0.25%), aspartame (0.8%), sorbitan monopalmitate (0.7%) is
prepared in an OLSA-type planetary mixer. The pulverulent mass is
dry mixed for 30 minutes and then supplemented with water in an
amount of 38 g per 100 g of dry mass, and blended for 1 hour at
room temperature. The homogeneous suspension thus obtained is
distributed into 1.5 cm.sup.3 polyvinyl chloride cells. The
cellular sheet containing the suspension is introduced into a
freeze-dryer immediately after division, to be frozen at a
temperature of -45.degree. C. under atmospheric pressure, for about
3 hours. Following this step the composition is heat-dryed for 12
hours under reduced pressure at a negative temperature, the
temperature is reduced to 30.degree. C. in steps of 5.degree. C.
The freeze-dried unit, extracted from the cell, is treated in a
fluidized bed (GLATT GPC-15) with injection of air (100 liters/min)
with a solution of hydrogenated castor oil (1 part), ethyl
cellulose (2.5 parts), beeswax (3 parts), cetyl alcohol (1 part),
groundnut flavor (0.15 part) in methylene chloride (80 parts).
Example 34
Cytomegalovirus Retinitis Treatment
[0190] Six patients with deteriorating eyesight due to CMV or
cytomegalovirus infection took the vaccine of the invention for 2
weeks. Every patient reported improved and reversed symptoms of the
disease. One patient who was blind and not able to see is now
capable to count fingers and to see shades of light.
Example 35
Weight Gain
[0191] The composition of the invention is given to over 40,000
patients with HIV. The majority of them experience weight gain. The
average weight is about 3 kg over 6 months. The increase in body
weight is statistically significant (p=0.008). More emaciated
patients however gain considerably more weight than patients who
are so wasted. This example indicates that vaccine of the restores
normal function of the gut and mucosal immune function.
Example 36
Quality of Life Improvement
[0192] About 400 patients' letters who request more supply of the
vaccine are analyzed. Over 85% of patients notice significant
improvement in their life quality. About 9% experience transient
adverse effects and 6% have no noticeable changes. The most common
reported symptoms include enhanced energy, ability to eat or
improved appetite, ability to walk among those who are bed-ridden
initially, ability to return to work, lack of headache, skin sores,
oral thrush, etc.
[0193] The entire content of patents, references, and internet
sites cited throughout this application are expressly incorporated
herein by way of reference.
[0194] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
scope and spirit of the invention.
* * * * *
References