EP0678033A1

EP0678033A1 - Human aids vaccine from revertant cattle virus

Info

Publication number: EP0678033A1
Application number: EP94903355A
Authority: EP
Inventors: Bhairab Chandra Bhattacharya
Original assignee: BHATTACHARYA FOUNDATION
Current assignee: BHATTACHARYA FOUNDATION
Priority date: 1992-11-25
Filing date: 1993-11-24
Publication date: 1995-10-25
Also published as: EP0678033A4; WO1994012212A1; AU5732594A

Abstract

A vaccine is provided containing a revertant Bovine Immonodeficiency Virus (BIV) from the retrovirus family, which exhibits an increased level of reactive ability and protectivity in humans as compared to the antibody producing antigen vaccines, the revertant BIV of the invention being exemplified by inducing clonal lymphocytes as immune surveillance against virulent retrovirus including HIV-1 and HIV-2.

Description

HUMAN AIDS VACCINE FROM REVERTANT CATTLE VIRUS

Field of the Invention

The present invention relates to cytotoxic T lymphocytes

(CTLs) and more specifically to vaccines comprising a revertant cattle virus derived by inoculating Bovine

Immunodeficiency-like virus (BIV) in calves that is characterized by producing cytotoxic T-lymphocytes (CTLs) in humans, the CTLs so produced processing super immune surveillance and killing power to dissolve virulent and revertant retrovirus infected cells in the body. The revertant of the . invention are exemplified by genomic molecular arrangement with , vaccinia and Marek's virus im unogenicity suitable to be formulated into an effective vaccine.

Background of the Invention

AIDS vaccine research, heretofore, has been targeted mostly to the production of antibodies against envelope protein-/fractions of "Human Immunodeficiency Virus" (HIV) antigens which are seriously mutation prone, with the search for an agent to enhance cellular immune response against HIV infection being still in its infancy. A number of unsuccessful attempts have been made to produce a vaccine against AIDS including, for example, formalin killed SIV and genetically altered cowpox virus, both tested in chimpanzees. Most vaccines against virulent viruses work by stimulating the body's humoral immunity producing antibodies which react with the virus' surface glycoprotein and Natural Killer (NK) cells, killing the virus with the help of antibodies. In this connection, Jonas Salk has proposed to immunize seropositive HIV individuals with HIV seropositive sera containing an antibody that presumably inhibits the function of Reverse Transcriptase enzyme (RT) , but such efforts have not been effective (Jonas Salk, Prospects for the Control of Aids by

SUBSTITUTE SHEET Immunizing Seropositive Individuals, Nature. 1987, v327, p473) .

Gonda et al. have determined and commented on the antigenic and genetic similarities of the "Bovine Immunodeficiency Virus" (BIV) to HIV, and suggested BIV's relevant candidacy for the viral intervention strategies applicable to AIDS. The suggestions were strictly limited to the extent of humoral antibody production by BIV in the human body and having an antigen-antibody reaction with HIV (Gonda, M.A. , et al.. Characterization and Molecular Cloning of a Bovine Lentivirus Related to Human Immunodeficiency Virus, Nature, 1987, v330, p388). The fact that antibody response to BIV proteins in humans is insignificant, however, has been shown by HIV Western Blot testing with human sera when sera from two laboratory workers who handled BIV in concentrated forms pricked themselves accidentally with infected needles and were contaminated. During regular examination, these workers tested negative for antibodies to BIV (Whetstone, C.A., et al, "Examination of Whether Persistently Indeterminate Human Immunodeficiency Virus type 1 Western Immunoblot Reactions are due to Serological Reactivity with Bovine Immunodeficiency-like Virus", Jour. Clinical Microbiol. , 1992, v30 (4), p764). The test also supports the nonzoonoses character of the cattle retrovirus BIV. The body's immune response against any foreign protein follows only two distinct courses of action: (1) Humoral response producing antibodies against the protein and (2) cellular immune response producing cytotoxic T-cells, natural killer cells or phagocytes to react instantly with the invading foreign protein and assist the humoral antibodies. Cytotoxic T memory stems are reactivated only at the time of reinfection.. In the absence of any antibody response and lymphocyte proliferation against BIV, as shown in the two human cases, the only remaining alternative is the presence of dormant cytotoxic T memory stems in the human hemopoietic system against revertant BIV. It can be activated by BIV or BlV-cross reactive antigen from a highly diverse strain of the virulent or revertant retrovirus.

SUBSTITUTE SHEET Cellular response against HIV, indeed, starts when people are first infected with HIV. Their immune system bursts into action, flooding the body with blood cells, the natural killer cells, and the cytotoxic T-cells. The natural killer cells attack other cells infected with the virus, as well as antibody-coated HIV which circulates in the hemopoietic system. When a person becomes infected with HIV, all viruses found in blood samples initially look the same. A year later there are six to eight different strains found in blood samples and after about three years, twenty different types of HIV may be found in the blood of the same persons (Cromie, W.J., Researchers Contribute to the Development of Vaccines and New Therapies to Slow the Advance of AIDS, Harvard AIDS Institute Suppliment. Fall 1992). Since the HIV vary from geographical region to another, it is unlikely that a multiple strain AIDS vaccine producing humoral antibodies has any real merit to it. In the distant future it is expected that HIV, like BIV, will become a revertant retrovirus by going back to the wild form. Unfortunately, the cytotoxic T-cell response against HIV breaks down soon after infection by a genetic variation in the Gag CTL epitope regions which allows the escape of cytotoxic T-cell recognition (Phillips, R.E., et al., Human Immunodeficiency Virus Genetic Variation That Can Escape Cytotoxic T Cell Recognition, Nature, 1991, v345, p453). Therefore, all attempts to produce a vaccine against HIV viral glycoprotein have failed to date.

No revertant vaccine like Marek's disease for chickens, is available for the protection of animals against pathogen retroviral infections such as visna and progressive pneumonia viruses of ship, equine infectious anemia virus, caprine arthritis encephalitis virus, simian immunodeficiency virus, feline leukemia virus, feline immunodeficiency virus and bovine immunodeficiency virus of cattle.

Summary of the Invention

I have now discovered a novel vaccine against pathogen retrovirus infection derived from BIV. The BIV by nature has

SUBSTITUTE SHEET become a revertant virus characterized by significant mutation, replication and revertant to a wild form as nonpathogenic and nonzoonoses. The protective ability of single BIV isolate containing numerous mutated forms, as compared to the antibody producing viral antigen vaccine, is significant. Cytotoxic T-cell memory stems against numerous point mutants and genomic variants of BIV, are capable of superimmune stimulation producing a series of novel cytotoxic T-cells against any virulent retrovirus including HIV-1 and HIV-2 which causes AIDS in humans. The class of cytotoxic T- cells so produced for multiple determinants on pathogen retroviruses is monospecific for a single determinant on all retroviruses and contains no other antiviral immune globulin, in contrast to prior art glycoprotein or core protein antibodies which are not monpspecific for any point mutant retrovirus.

It is accordingly, one object of this invention to provide more effective therapeutic treatment during the course of retrovirus infection with BIV inoculation by virtue of which there is an improved process for the clonal production of a particular set of lymphocytes which will give cellular immunity against retrovirus infection as described previously.

A further object of this invention is to provide instant cytotoxic T-lymphocytes against HIV which destroys T4 cells in the body and point mutants of HIV which escape immune surveillance.

Another object of the present invention is to provide immunity to individuals against exposure to AIDS viruses, by vaccination with BIV and producing cytotoxic T-memory stems against HIV point mutants.

It is a further object of the invention to provide a method for preparing nonvirulent BIV for inoculation in animals, such as cattle and humans. A still further object of this invention is to provide a therapy for treating individuals having AIDS, by inoculating higher doses of heterogeneous BIV isolate.

SUBSTITUTE SHEET It is a still further object of the invention to provide an essentially heterogeneous isolate of the BIV for vaccination against any pathogen retrovirus.

Other objects and advantages of the invention will become readily apparent from the ensueing description.

Description of the Preferred Embodiments

In satisfaction of the foregoing objects, there is provided by this invention a novel composition of nonvirulent revertant BIV which produces cytotoxic T-lymphocytes in vivo, combating with another retrovirus such as HIV-1 and HIV-2 in humans, the cytotoxic T cells themselves, as well as a vaccine and therapy employing the revertant BIV isolates. In one aspect of the present ijivention, BIV is propagated in young calves as follows: Following inoculation of BIV contaminated cells or whole blood in calves, they are watched for three to four weeks and then the young calves are inoculated with a booster shot. After four weeks of the initial inoculation, one quart of blood is aseptically obtained every three weeks, syncytia forming white blood cells are separated,, the cells are disintegrated in physiological saline and then filtered through 0.2 micron millipore filter for viral isolates. From about 100 to 500 million lyophilized BIV particles/dose is suitable for vaccination against a pathogen retroviruses infection such as HIV-1 for permanent immunity.

The course of smallpox vaccine immunity in humans by cowpox producing vaccinia virus has some parallel with the present revertant BIV vaccine described herein. The vaccinia virus contains two double-stranded large DNA strands with each end covalently sealed through phosphodiester-linkage. The peculiar ends of these DNA molecules evolved because of the difficulty in replicating the last few nucleotides at the end of a DNA chain (The Molecular Biology of the Cell. Bruce Alberts et al. Edited, 1st Ed., Garland Publishing, Inc. N.Y., 1983, p233). The double-stranded DNA of the poxvirus replicates using DNA dependent DNA polymerase, which under

SUBSTITUTE SHEET stress, produces severe mutants within a single isolate. Because of its revertant character, it is successfully used as a vaccine against smallpox infection, facilitating production of numerous cytotoxic T-cell memory stems in the body. An early observation of great importance was that instead of humoral immunity, smallpox vaccination produced cellular immunity, provided by CTL, against the smallpox virus. It is supported by the fact that many agammaglobulinemic patients, when immunized with the smallpox vaccine produced cellular immunity to prevent smallpox outbreak. The agammaglobulinemic patient receives lifelong immunity against smallpox because of the presence of a cellular immunity. Edward Jenner invented smallpox vaccine (in Latin, the word Vacc means cow) , in 1796, observing that the cowpox virus was non-pathogenic to humans and, at the same time, produced immunity in milkmaids against the smallpox virus. He was so sure of his theory that he immunized an eight year old boy with cowpox-producing vaccinia virus. He then held a public vaccine exhibition to show the effectiveness of the cowpox virus against the virulent smallpox virus.

The hypothesis that the immune response to cattle vaccinia virus and the BIV are cellular rather than humoral, is also supported by the response of agammaglobulinemic children to BCG immunization. The children develop typical delayed skin reactivity to tuberculoprotein. Such patients can be sensitized by dermal contact with 2.4-dinitro- flurobenzene and by intradermal injection of diptheria toxoid or horse serum precipitated with horse antitoxin or rabbit anti-horse serum. The delayed sensitivity can be transferred to normal individuals by subcutaneous injection of the agammaglobuliinemic patient's leukocytes, but not by even large doses of serum (Carpenter, P.L., Immunology and Serology. W.B. Saunderss Company, London, 2nd Ed., 1965, p358). The clonal lymphocytes against unseen epitopes of the BIV and vaccinia viruses are linked to the same origin in the lymphoid tissue in the body (Bhattacharya, B.C., Cattle AIDS, Proc. Zool. Soc Calcutta, 1992, v45-l, p.l).

SUBSTITUTE SHEET The genetic organization of BIV is most similar to that of other pathogen retroviruses. However, Garvey et al. identified a series of Alu-1 sequences in the LTR,near the TATA Box, as shown below. The presence of a series of Alu-1 restriction enzyme recognition sequences surrounding the BIV cap sites creates numerous point mutations and displays extensive genomic variation within a single BIV isolate (Garvey, K.J., et al., Nucleotide Sequence and Genomic Organization of Biologically Active Proviruses of the Bovine Immunideficiency-like Viruses, Virology. 1990, vl75, p391).

U3

TATA Box

,Alu....Alu...Alu.Alu.

+4 +31 cap site at the U3-R boundary with repeated Alu recognition sites ( Garvey et al. Virology. 1990, vl75, p400 ).

A comparison of the sequence and locations of the Alu-1 sequences in different mammals suggests that these sequences have multiplied to high copy numbers relatively recently and they are scattered throughout the genome. The function of the Alu-1 sequences in BIV genome, to date has been obscure. It is revealed now that these repeated motif of Alu-1 sequences surrounding the BIV cap site is useful in providing structural conformation for regulation of transcription. Unlike other retroviruses, a single isolate of BIV produced in vivo, is highly heterogeneous. One possible reason of this genomic variation is the inherent infidelity of the retrovirus reverse

SUBSTITUTE SHEET transcriptase enzyme (RT) . During reverse transcription of the viral RNA, the enzyme RT constrains the secondary structure of the viral RNA, which stems from the Alu-1 repetitive sequence around the cap site in the BIV genome and makes the RT even more error prone. This event reflects ultimately in numerous mutations in the core protein of the virus until is a revertant virus.

The revertant BIV can produce an effective vaccine following the revertant character of a virulent virus which has successfully been used as a vaccine against Marek's disease caused by herpes virus in chickens. (Witter, R.L., Revertant Serotype 1 Marek's Disease Vaccine, U.S. Patent Number 4,895,717, Jan. 23, 1990).

A phenomenon that would lead one to suspect the significance of BIV in connection with the treatment of human patients infected by HTV-l and HIV-2 has been observed in India. Cows are considered to be "holy" in India, and many prostitute abodes in large cities, such as Calcutta, and small villages, such as Kuku Ka Bas, are allowed to keep cows, or are dependent on dairy cattle, as a symbol of wealth and prosperity. This might have influenced the slow spreading of AIDS among Calcutta prostitutes (approximately 0.07%) compared with the high rate of HIV infection (16.0%) among prostitues in Bombay where no cows are allowed in the "Red Light Zone." In some of the tribe's villages in India, prostitution is a traditional way of living together with dairy farming and agriculture. AIDS is unheard of there (Philip, S., After year of Denial , Asia Faces Scourge of AIDS, The New York Times, Sunday, Nov. 8, 1992, pi; Gargan, E.A., Tribes's Village in India is Supported by a Bordello, The New York Times, Sunday, Nov. 15, 1992, p. 14: and Bhattacharya, B.C., et al.. Preparation for AIDS/HIV Vaccine Evaluation in India, NIAID Re: 1 R21 Al 33895-01 ARC, Sept. 3, 1992)

The Indian cattle are heavily infected with BIV. Daily contact with infected cows and drinking infected milk results in a natural BIV infection among the prostitutes who are apparently protected from AIDS even though, they have come in contact with HIV during their commercial sex life.

SUBSTITUTE SHEET The course of HIV infection prevention, using revertant BIV protein related cytotoxic T cell response in vivo, as described herein, is believed to represent an advancement in knowledge relating to the field of AIDS vaccine research. In an effort to avert any large scale outbreaks of AIDS in the future, the search for improved vaccines has continued.

Although the term "vaccine" is properly reserved for smallpox prophylaxis in humans, the term is not improper in the case of BIV since it originates from cattle and will be used to protect humans from HIV infection. So, Vaccine is defined herein in its broad sense to refer to any type of biological agent in an administrable form capable of stimulating a cellular response in an animal or humans inoculated with the vaccine. Attenuated organisms are safe immunizing agents if they do ,not produce any pathological symptoms in the host species. BIV is known to be nonpathogenic to humans.

The term "revertant" is intended to refer to BIV having numerous point mutations in the core protein and characterized by, after significant replications, going back to a nonpathogenic wild form, as compared to the attenuated form where virulence is minimized by animal passage. The term is used herein generically to encompass both true revertants and apparent revertants from a population of viral particles rather than from a cloned virus.

In accordance with the present invention, the method for treating a human patient against a retrovirus infection comprises preparing a single BIV isolate with multiple epitopes: in vivo transfer of the BIV isolate: production of heterogeneous population of CTLs against different core protein epitopes; recognition of heterogeneous retrovirus epitopes by BIV induced CTLS; and protection of retrovirus infection by way of a vaccine or chemotherapy.

Appropriate adjuvants as shown in the art may also be included in the vaccine formulation. In particular cases, the vaccinal efficacy can be enhanced ny combining the revertant BIV with other viral agents into bivalent or polyvalent vaccines.

SUBSTITUTE SHEET EXAMPLE I

Production of BIV in Live Calves (Nonvirulent Revertant BIV Vaccine)

Nonvirulent BIV from vector cattle, is the original source of the BIV vaccine. Typically, the vaccine will contain at least about 100,000 PFU (plaque-forming units) or syncytia of the virus, and preferably between 200,000 and one hundred million virus particles. A larger quantity of BIV absorbed in aluminum sulfate may be used for treatment or prophylaxis against HIV in human.

Three calves (5-7 months old) are inoculated intravenously with approximately 200 ml of blood from a BIV infected 9 year old cow. The cow has lost her last 4 calves, two to three days after delivery. She is showing progressive emaciation, milk loss and swollen subscapular, prescapular, lumberfossa and mandibular lymph glands. These symptoms are considered to be typical for the BIV infection. A sample of blood in 0.2% sodium citrate solution showed syncytia formation of lymphocytes under the microscope, confirming the BIV infectivity.

The calves are further inoculated after 4 weeks with the same blood drawn from the BIV-infected cow. Two hundred ml of blood is collected through puncturing asceptically the jugular vein of each calf, in a sterile glass cylinder containing 0.4gm sodium citrate, to prevent clotting. Samples are stored in 200 ml glass cylinders for formation of buffy coat with leukocytes. After overnight standing, the buffy coat is removed in a petri dish for checking units of syncytium/ml of blood, under an inverted microscope. The buffy coat samples are disintegrated by homogenization and diluting with a required amount of sterile saline. The sample is then passed through 0.2 micron millipore filter. Filtrate containing approximately ten million particles per ml (one million per syncytium count), is lyophilized and stored in sterile vials for subcutaneous inoculation in humans.

SUBSTITUTE SHEET EXAMPLE II Human Test with Revertant BIV Inoculum Inducing BIV Cellular Response against HIV Infection:

To find a covariation between a natural BIV infection and its influence against HIV exposure to a high risk group of prostitutes in India, twenty samples of blood from individuals who visited a Humanity Association of India Clinic (HAIC) , Calcutta, between 1991 and June 1992, were tested. The prostitutes were commercial sex workers who lived in the same prostitute abode which kept BIV+ milk cows. Most of the prostitutes visited the clinic for complaints against sexually transmitted disease. They were considered to be in a higher risk HIV+ category. All blood samples were tested for HIV+ with an Elisa test at the Institute for Tropical Medicine (ITM), Calcutta. The Institute keeps an up-to-date record for all prostitute blood tests done there. Only the HIV+ samples are sent out for Western Blot test for further confirmation. The center provided a record of 2,000 individual tests with 30 Elisa positive and 12 of them confirmed Western Blot positive cases, up to June 1992.

The twenty samples tested for a covariation analysis of HIV+ vs. BIV+, against 2,000 random samples, showed all to be free from HIV antibody, which compared favorably with a prevailing finding of 16% positive cases in Bombay for patients who were not subject to BIV. This study contributes to the hypothesis of the protective action of BIV related CTLs against HIV infection. See Table 1.

Using vaccine prepared as described in Example I, will provide a similar or better protection against HIV infection amongst a population of patients who are not living in an atmosphere of BlV-infected cattle.

SUBSTITUTE SHEET * Western Blot Test: Elisa+ samples from ITM Maharastra (+Boιnbay) W. Bengal (+Calcutta )

* Source: AIDS Control Organization, New Delhi ( Bhaumik, S, Myanmar as AIDS Bridge, India Abroad Nov. 20, 1992 ) .

It is understood that the foregoing detailed descriptions have been given merely by way of illustration and that modifications and variations may be made therein without departing from the spirit and scope of the present invention.

SUBSTΓTLΠΈ SHEET

Claims

1. A vaccine for treating a patient against a pathogen retrovirus, which comprises a revertant BIV isolate having numerous point mutations in the core protein and characterized by after significant replications, going back to a nonpathogenic wild form.

2. The vaccine for treating a patient against a pathogen retrovirus as claimed in Claim I, wherein said revertant BIV vaccine is adopted to be administered to a HIV positive patient to be treated in an amount of 200,000 to 100 million virus particles per dosage, sufficient to induce super immune stimulation for said patient to produce a clone cytotoxic T lymphocyte reponse which will kill all HIV infected cells in the circulation and release toxic chemicals to dissolve the cell genome containing HIV.

3. A method for treating a patient against a pathogen retrovirus comprising administating an effective immunization dosage of revertant BIV vaccine as claimed in claim 1. to a healthy individual to be treated wherein said vaccine is administered in an amount sufficient to induce superimmune stimulation to produce by said individual a clone cytotoxic T lymphocyte which will kill retrovirus infected cell in the circulation, by direct contact and release toxic chemicals to dissolve the cell genome containing retrovirus.

4. The method for treating a patient, as claimed in claim 3, wherein said revertant BIV vaccine is administered in an amount of from about 200,000 to 100 million virus particles per dose, an amount sufficient to kill HIV-infected cells in the circulation and releasing toxic chemicals to dissolve the cell genome containing HIV.

5. A vaccine for treating an animal including cattle, against a pathogen retrovirus, which comprises a revertant BIV having numerous point mutations in the core protein and characterized by after significant replications, going back to a nonpathogenic wild form.

SUBSTITUTE SHEET