WO2002007760A2 - Therapeutic agent against aids comprising anti hiv goat antibody - Google Patents

Abstract

A composition is provided for the treatment of HIV and AIDS, the composition comprising antibody of a goat after challenge with a human HIV virus, and a method for treatment of a human infected with the HIV virus comprising administration of the antibody to the infected individual.

Description

Therapeutic Agent

The present invention relates to therapeutic agents for treatment of disease, in particular for the treatment of HIV.

BACKGROUND OF THE INVENTION

Acquired immune deficiency (AIDS) is epidemic in many areas of the world. The disease is considered to be caused by HIV, Human Immunodeficiency Virus, which exists in different forms. In addition, the SIV virus known to infect macaques is thought to have a similar mode of action to HIV.

Reported cases of HIV infections are still increasing world -wide. Clinical treatments, such as the combination protease inhibitor treatments, only represent minor improvements in the management of health care in patients with HIV infection.

There is still a need for an effective treatment for HIV, both in the prevention of HIV infection into cells such as CD4 positive T cells, and in elimination of the virus from the body.

The present invention sets out to address this need. SUMMARY OF THE INVENTION

In the first aspect, the present invention relates to a composition which can be used for the prevention and treatment of HIV and AIDS, the composition comprising antibody, preferably the total antibody population, of a goat after challenge with human HIV virus.

In general, injection of antibodies into humans derived from a non- human host is counter-indicated. A strong immune response is normally mounted against the foreign antibodies themselves. However, surprisingly, we have discovered that use of goat serum extract does not provoke the immune reactions which are anticipated with other foreign animal proteins. Injection of goat antibodies is tolerated both by immunosuppressed patients and normal individuals. We have also discovered that the goat antibodies is then able to block entry of HIV into cells and to kill HIV infected cells.

Thus, the reagent of this invention is a composition of the antibody population from the blood of a goat, following HIV challenge. SIV or other known or new viruses may be employed, provided that they elicit the formulation of an effective reagent. Preferably a cocktail of HIV immunogens is given, such as a cocktail of HIV lysates, though there are other options including live cells. A cocktail is a mix of at least two, preferably at least three, or more usually more than three.

Preferably the antibody population of this invention is one which can be obtained by a process involving raising anti-HIV or other effective antibodies in a goat, draining blood from the goat, demonstrating anti- HIV antibody neutralising capability in the drawn blood, removing solids from the blood, precipitating solids using supersaturated ammonium sulphate or other suitable precipitation agent, separating the precipitate, dissolving the precipitate in a suitable aqueous medium, and dialysing the solution for example with a cut-off of 5 to 50,000 Daltons, preferably 7 to 30,000 Daltons, more preferably 8,500 to 15,000 Daltons, especially about 10,000 Daltons.

In particular, the invention provides a composition including the active component which can be derived from the blood of a suitably challenged goat by a serum extraction technique that is not designed to isolate individual, specific antibodies. In particular, the invention envisages isolation of the active component, possibly a mixture of cooperating molecules, from blood serum of the challenged goat, without exhaustive purification and extractraction to obtain an individual antibody.

The present invention preferably uses the total population of antibody molecules, derived from HIV challenge to a goat. Without wishing to be constrained by theory, we believe that such an approach possesses significant benefits. Patients treated with such a total antibody population showed significant benefits within minutes of being treated. Such a response is akin to successful treatment of a toxin. In some way, the total antibody population derived from a goat which has previously been infected with HIV acts to suppress 'toxin-like' effects caused by HIV infection, as well as effects of HIV on populations of immune cells such as CD4 positive T cells. In addition, the goat has antibodies to agents which cause many of the common opportunistic infections contracted by an AIDS/HIV patient, providing additional protection. We have also observed a beneficial effect of the total antibody population raised to HIV on diabetes and multiple sclerosis in treated individuals.

Preferably, the composition is purified and consists essentially only of antibodies, or comprises a purified total antibody preparation.

Purification of the antibody preparation of goat after challenge with virus such as HIV may be carried out by any suitable means. Preferably the purification is carried out using the method exemplified in Example III. The present invention also extends to antibodies made by the method of the invention.

In a further aspect, the invention relates to a method of preventing HIV infection or treating an individual infected with HIV, comprising the steps of

(1) exposure of a goat immune system to HIV;

(2) purification of the total antibody population derived from the goat after HIV challenge; and

(3) treatment of the individual with the antibody preparation obtained in step 2 above.

Goat antibodies produced as described herein may be formulated in accordance with the invention in a composition to inhibit viral replication in vitro or in vivo. As such, the invention also relates to pharmaceutical compositions comprising the goat antibodies of the present invention, suitable for the treatment of disease, such as viral disease. The ajitibodies of the present invention may be mixed with suitable pharmaceutically acceptable carriers.

Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) with suitable composition, or oral, topical or parenteral administration, and they may comprise a carrier. The compositions may need to be sterile when administered parenterally.

Administration of the composition of the invention may be by any suitable method such as by intravenous infusion, subcutaneously, intra-muscular injection, oral preparation, intraperitoneal and intravenous administration. The correct dosage will vary according to the particular formulation, the mode of application, and the particular situs, host and condition being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivity and disease severity shall be taken into amount. Administration can be carried out continuously or periodically within the maximum tolerated dose.

The composition of the present invention may be used with other drugs to provide a combination therapy. The other drugs may form part of the same composition, or be provided as a separate composition for administration at the same time.

Treatment is given by means of a subcutaneous injection, in amounts varying between one and ten ml and is designed to deliver the medication as speedily as possible to the lymphatic system. Treatment is normally of three months duration and following the first inoculation most patients evince the following:

• Within 20 minutes became substantially more alert, with a growing feeling of well being.

• Within 80 minutes start to regain their normal pre-infection appetite.

• Within 2 hours sought to undertake physical exercise such as swimming or a shopping expedition.

• Within 60 minutes became voluble and appeared to have recovered from their prior moderate to severe depression.

Continued treatment yielded the following benefits:

1. Within approximately 2 weeks of the first treatment the patient started_to gain weight.

2. Laboratory reports confirmed that 4 to 6 weeks after the first treatment, viral loads and P24 values were dropping substantially and CD4 together with other helper T cells were returning to normal levels.

3. The product was well tolerated and effective without any apparent side effects. 4. No evidence of serum sickness, localised skin reaction or anaphylactic shock, was present.

In summary, a full course of treatment (varying between 8 and 12 weeks, which is dependent upon the clinical condition of the patient) resulted in: -

5. A proliferation of helper T cells being generated, thereby returning the CD4 and CD8 cell count to within normal ranges.

6. A decrease in the patient's viral load to theoretical zero, generally in increments of 0.5 logs or greater.

7. A reduction of P24 values to zero.

It is important to note that the present medication, unlike current treatments, does not require the patient to maintain a strict hourly or daily regime and relies (depending upon clinical condition) upon a simple injection being administered either daily, weekly or monthly.

A test dose is employed usually to see if the person develops an allergic reaction to the hyperimmune goat serum. We use 0.1 ml intradermally, wait approximately 30 minutes to see if we get an intermediate reaction. If this reaction is negative, then the assumption is that an immediate sensitivity reaction is most likely to occur..

The intent is to get the dosage up to at least 0.3 ml per hour for a 24- hour span, which is approximately 7 ml in a day. We would continue this process under close observation for at least seven days. The rationale for this is that we know that a dilution of 1-20 over a seven- day incubation period in vitro rendered complete elimination of cell fusion with the HIV virus with the human CD4 cells. So, this is our starting point. The amount of reagent actually needed may not even need to be that much, or it may need to be a little bit more.

The invention also extends to a method of generation of a protective composition comprising antibodies for use in protection of a non goat species, the method comprising immunising a goat with a non goat antigen (e.g. a virus or foreign protein), and purifying the total antibodies extract produced in the goat after challenge with the antigen. The total antibodies population may then be used to protect the non goat animal from the antigen used as immunogen.

Suitably, the antibodies produced in the goat are tested for protective activity prior to use as a protective agent, through suitable in vitro or in vivo assays, such as those of Example II.

The present invention further relates to use of a composition comprising antibody of a goat, preferably the total antibody preparation of a goat, after challenge with a human HIV virus in medicine, and the use of a composition comprising the antibody of a goat after challenge with a human HIV virus in the preparation of a medicament for the treatment of HIV and AIDS.

Preferably, the composition of the present invention is rendered sterile by one or all of the following: precipitation with about 45% (47% might also be used) ammonium sulfate, freezing at -70°C for 24 hours or microfiltration.

The invention extends to the isolated active component as well as mixtures including the active component.

SPECIFICATIONS

Animal Husbandry Procedure

In describing the animal husbandry procedure, the animal is housed on a private farm of a veterinarian. The particular animal is isolated out from the herd itself. Being isolated out from the herd, this animal is initially examined and then shown to be physically normal and absent of any microbiological illnesses, included CAEV, for which the animal is tested to make sure that it is CAEV negative.

The HIV immunogen

Production of immunized serum used in the invention:

Animal requirements:

For purposes of the invention, a goat's immune system was used to make the immunized serum. The same results would be expected in other species immunized with the immunogen. Though any healthy goat's immune system would produce an immune response, the ideal animal would be an animal greater than 4 months of age, larger than 30 kg, and a castrated male or barren female.

Immunogen Requirements and preparation:

HIV3b.sub.MN, HIVl .sub.BAL were purchased from the Scripps Institute San Diego in the form of heat killed purified viral lysates. Analysis of these purified viral lysates demonstrated lot to lot variation in total protein content with a range of l.Omg/ml to 1.2 mg/ml.

HIV-1 isolates: 91US056, 92HT593, 92US723, 92US657, 92US660, 92US714 in a viral cocktail form were supplied by the United States National Institute of Health, (NIH) in the form of heat killed purified viral lysates. Analysis of these purified viral lysates demonstrated lot to lot variation in total protein concentration of 0.556 μg/ml. (10.4 μg total protein in 18.7 ml)

In both cases the immunogen used is a killed viral lysate, those familiar with the art would concur that a live virus would have the same effect because both contain the same proteins and peptides of interest, and that killed virus reduces the risk of live viral exposure.

The specifications for the immunogen is that all of the immunology, isolation, preparation, serotyping is performed by an immunologist that works on AIDS research. The immunologist uses a macrophage trophic strain of HIV which is prepared by growing the virus in a CEMX174 cell; that is Sherma, et al. (Derivation of neuro trophic, lympho trophic, parental virus: pathogenesis of infection in macrophage) Journal of Virol 66: 352-356 (1992). The CEMX175 cell is immortalized CD4 bearing human TB hybrid cell lines that is highly susceptible to HIV- induced cytopathicity or fusion, and passive for replication by HIV, see Hoxie, et l., (Biological characteristics of a simian immunodeficiency virus like retrovirus (HTLV-IV) : evidence of CD4 associated molecules required for infection) , General Virology 62: 2557-2568 (1988) .Koenig, et al. selective in section of human CD4 plus cells by HIV simian immunodeficiency virus: productive infection associated with envelope glucoprotein induced fusion.

After meeting the acceptance specifications of the immunogen that is used in the goat, one million particles are then grown per ml of solution. At that point in time, the immunogen is then heat killed at 60 degrees for 30 minutes and then quick frozen in a -70 degrees freezer and stored and shipped in a frozen container. It is then heat killed once more. Once it is heat killed once more, 60 degrees for 30 minutes, it is then injected one ml with one million particles every week for the next four weeks, and then at six weeks the animal is then bled to obtain antibodies.

Safe Handling Immunogen

All of the virological handling of the live virus is under the direction of the virologist. The usual sero-techniques using at least a P3 laboratory is used to prevent infectious organisms from being transmitted to humans. Manufacturing Process

The virus is grown in a petri laboratory by a certified virologist who not only grows, but serotypes the virus, and then maintains live virus for future fusion studies to identify the presence of neutralizing antibodies that have been produced in the goat after it has been immunized with the HIV virus. Before shipping the virus, a heat treatment kills the virus to 60 degrees for 30 minutes. The killed virus is then quickly frozen to -70 degrees C, and then put it in a cold container to keep the virus frozen. After the virus is received at the laboratory, the virus is then heat killed once more by the immunologist. 1 ml of the virus is then utilized and then the remainder is frozen.

Amount of immunogen:

Those familiar with the art will agree that the amount of immunogen can vary widely and still produce a positive immunogenic response.

The amount used was derived from previous experience with both immunogens. For example, the 5 HIV-3B goats received 1 mg of protein for each of the first two immunizations. This was reduced to 200 mg/goat per for the following two immunizations.

The 6^th goat (NIH cocktail goat) was immunised with a total of ~2 mg (micrograms) over two immunizations.

Both groups of animals produced an immune response to the foreign proteins. Adjuvants:

The addition of adjuvants is recommended to enhance the immune response. Though it is possible to elicit an immune response without an adjuvant, it is advisable to use one. Many adjuvants are commercially available. We have used both Freunds adjuvant and RIBI for the purpose of the invention.

Methods of administration:

Administration of the immunogen (with or without) an adjuvant can be done by injection. Injection routes include subcutaneous, intramuscularly, peritonealy, or intravenously etc. Administration can be a one time injection, or a series of injections that are usually between two to four week intervals.

For preparing the immunization using RIBI adjuvant, we used a category II biologies hood. The immunogen and RIBI were emulsified together as per the protocol accompanying the RIBI. The amount of immunogen used ranged between 200 ug/ immunization to 1000ug/ immunization. Sterile saline or lx PBS (pH 7.4) was added (if needed) to create a total volume 1ml of immunogen per injection. Once mixed, goats numbered 0125, 0126, 0127, 0128 & 0129 were given the immunogen intramuscularly.

Goat number 0378 was given Freunds adjuvant. This was prepared in the hood using a 1: 1 ratio of viral lysate cocktail immunogen solution and Complete Freunds adjuvant followed by Incomplete Freunds adjuvant. This immunogen is administered by any one of a variety of injection method (subcutaneous, intramuscular etc). The amount of immunogen used for the invention was less than 2 μg per immunization. We immunized the goat intradermally at 20 sites with a volume of 50 μl/site.

Every week for the next four weeks, then the animal is actually injected, which has been examined by a certified veterinarian to make sure the animal is in good health, the animal is free of any microbiological infections or any physical defects, the animal is tested for CAEV, and all animals used are CAEV negative, and preferably female goats that are lactating.

Once the animals have been injected over the four- week period, and have also been isolated unto themselves and fed in the usual manner with hay, oats, water, the animal is then injected at approximately six weeks for a booster of the immunogen which is the HIV virus, and then at approximately seven to eight weeks the blood is then drawn from the animal to test it for the presence of neutralizing antibodies. The animal is bled. The blood is then transported for tests for the presence of neutralizing antibodies to make sure that fusion is prevented. One uses the usual steps in showing that the CD4 cells have been shown to have no fusion sites present, in the usual titration manner, with increase inthe concentration of the serum to the point that produces no fusion. This information is then faxed or transported back to the immunologist, who lets us know that we do have neutralizing antibodies present that do present cell fusion. Collection of whole blood and the processing of it for serum.

The serum demonstrated activity after 42 days, at which point the collection of serum commenced.

Blood is drawn from the jugular vein into a sterile blood bag without anticoagulants. Standard blood bag sizes range between 100 ml- 1000 ml capacities. The blood is allowed to sit and clot for a period of time, usually between 8-24 hours at 4 C. After sitting, the serum separates from the clot containing most of the blood cells inside the bag. The liquid serum can either be decanted off at this point. To increase the serum yield, the blood bag can be placed in a centrifuge and spun. For collection of serum for the invention, we collected whole blood in either 500 ml or 1000 ml blood bags. These were stored at 4 C. for 18-24 hours and centrifuged at ~3500 g (3000 rpm with a 26cm Radius centrifuge rotor) for 15 minutes. The serum is then decanted from the bag into a centrifuge bottle and spun again at 3,000 rpm for 15 minutes. Once the second spin is complete, the serum is bottled and labeled.

Alternative methods of collection include collection of whole blood into plasma bags (blood bags with anticoagulants). Collection of the blood is through the jugular vein. Once collected, the blood bag is placed in a centrifuge and spun at ~7500 g for 35 minutes. (5000 rpm with a 26 cm radius centrifuge rotor) Once the blood plasma is separated, a plasma extractor is used to help decant the plasma from the blood cells at the bottom of the bag. At this point, the blood cells can either be discarded, or put back into the goat. The plasma can either be bottled and frozen, or turned into defibrinated pla.sma (serum) by adding 1% by volume of a 1 M solution of CaCtø (Calcium Chloride) and allow this to sit for 2 hours after being mixed. The resulting clot is chopped up and placed in a centrifuge bottle and spun at 7500g (5000 rpm with a 26 cm radius rotor) for 30 minutes. The resulting liquid is decanted and stored. The solid fibrin is discarded.

Defibrination (removal of fibrin) from plasma

Reagents: 1M CaCl₂

Procedure:

1. Pour plasma into glass containers or covered beakers.

2. Stir in with a rod 1% by vol. of 1M CaCl₂. Wait 1.5-2 hrs.

3. If plasma has yet to clot, stir in another (1% by vol.) of 1M CaCb.

4. Once solidified, cut the clot with a knife so one can easily pour the cut up clot into a 1 liter centrifuge bottle.

5. Place the cut up clot/ serum mixture into 1 liter centrifuge bottle.

6. Centrifuge for 35 minutes at 6,500 g, (temp: 15 C) in a Sorval RC- 3B or other large centrifuge .

7. Decant serum (defibrinated plasma) from centrifuge tubes

8. Measure volume, bottle, label and store at -20 or colder.

Procedure to produce an immune response from the HIV cocktail goats using Freunds Adjuvant. Materials:

1. Adjuvant

2. Hood

3. Immunogen

4. Sterile Saline and/ or lx PBS pH 7.4

5. 20 Gauge x 1" 3 ml needles

6. Stainless steel emulsifying needles for connecting syringe barrels

7. Marking pen for syringe identification

8. Sharps container.

Procedure:

1. Turn on the biologies hood. Wipe the inside with alcohol.

2. Turn on the germicidal light for at least 10 minutes

3. Remove immunogen form storage area and place in hood

4. Access how much buffer or saline is required to bring the total immunogen/ buffer solution to 0.5 ml.

5. Label a 3 -ml syringe with the proper animal number and draw up the required amount of saline required to make the 0.5-ml volume, (if needed)

6. Top up the syringe with the immunogen solution to make 0.5 ml.

7. Remove the needle and connect the barrel containing the solution to the emulsifying needles.

8. Take a second needle and draw up 0.5 ml of Freunds adjuvant. Remove the needle and connect the barrel to the other end of the emulsifying needle. 9. Emulsify by pushing the syringe plungers back and forth. This will mix the adjuvant with the immunogen. The result should be a thick emulsification.

10. Place the material into the syringe with the goat identification on, remove the emulsifying needle and replace with the original 20 gauge needle that was just removed.

11. Clean emulsifying needles and discard any contaminated materials in the sharps bucket.

Immunization and serum collection of goats using RIBI:

For efficient and an optimal immune response, immunizations should be done on a routine schedule. Traditionally, these would be at between 14-45 day intervals, but can easily be performed with greater or less frequency. Though different immunization cycles and amounts can be used.

The HIV 3B goats were immunized with 1 mg of HIV-3B viral lysate (Scripps Labs, San Diego, Ca) and RIBI adjuvant (MPL+TDM+CWS emulsion Sigma Chem. #M 6661). The schedule went as follows:

Day 0 Immunize 1 mg/HIV 3B lysate/ RIBI

Day 7 Immunize 1 mg/HIV 3B lysate/ RIBI

Production bleeds started at day 28 and continued on a 14-day cycle as a result of these immunizations. Serum was harvested via the collection and clotting of whole blood or through plasmapheresis. The goats received a second immunization a year later.

Day 380 Immunize 200 μg HIV 3B lysate/ RIBI Day 411 Immunize 200 μg HIV 3B lysate/ RIBI

Again, the animals were placed on a 14-day bleeding cycle. Again, the immunized serum was harvested via the collection and clotting of whole blood or through plasmapheresis.

The veterinarian then draws approximately 400 ml of blood from the goat under sterile technique. The vet shaves the area for needle extraction and then preps it with betadine. An 18-gage needle is used and draws approximately 400 ml. Of blood from the animal. Of note is that the animal can tolerate approximately 400 ml. Of blood drawn without the animal suffering any untoward effects. The animal does not have to be sacrificed. The animal can then be re-bled in approximately 10-14 days after it replenishes its blood volume. This blood is then transported under sterile technique in sterile containers to the immunologist. He then separates the serum from the blood cells by centrifugation. After the separation procedure is performed, he then uses a P2 hood and with micro-pipetting, he separates out the serum with micro-pipetting from the blood cells and places it in a sterile beaker. This serum is placed in a sterile beaker which has been microwaved in a microwave oven for approximately three minutes to insure that there is no microbiological organisms present. After placing the serum now in a sterile container from a sterile pipette from a sterile drawing of the blood from the animal, the serum is completely separated and then the serum is then titrated with supersaturated ammonium sulfate solution. Ammonium sulfate will selectively precipitate out the immunoglobulm within serum and leave all the other proteins and other products floating within the water content of the serum. After precipitation is completed, the immunoglobulm, or antibodies, are then isolated by centrifugation. They float to the bottom of the tube. After floating to the bottom of the tube, the supernatant liquid is poured off. Once the supernatant liquid is poured off and there is pure antibody present, a buffer is then added to the immunoglobulm. This particular buffer which is added to preserve the quality of the antibody is the usual buffer that is used in immunology.

While the animal is being injected during the immunization phase, the animal is given an adjuvant and the goat's immunogenic response is augmented by using MPLR (RIBI Immunochem Research, Inc.) plus TDM adjuvant system (Sigma Chemical Company, St. Louis, Missouri) according to manufacturer's instructions, i.e., administering intramuscularly 50 microliters into each hind leg.

After the immunoglobulm has been separated and a buffer is added, the dialysis process then takes over. The dialysis is performed in a buffer with a selective semi-permeable membrane that allows for the ammonium sulfate to be separated out now from the immunoglobulm. This process is continued for approximately 24 hours. A magnetic sterile plus the sterile beaker with the buffer solution is present while the dialysis process is taking place. This process continues again for approximately 24 hours until all of the ammonium sulfate has diffused out of the dialysis bag. Now we are left with pure immunoglobulm in the dialysis bag. This process is then repeated until no ammonium sulfate is present. At that point in time, the antibodies which have now been prepared are then quick frozen at a -70 degrees again and ready for transportation for microbiological examination and chemical examination. This particular solution is then tested for viruses, fungus, and bacteria by the usual microbiological cultural scheme, unless it is not used until a negative viral culture, negative fungal culture, and negative bacterial culture are present. Gram stain, AFB stains, and anti- fungal stains are all performed on this particular solution. This is still one lot of antibody which has been prepared. Of note is that the antibody is also analyzed for immunoglobulins. The immunoglobulins that are present within the serum are measured by using immunoglobulin electrophoresis. In this particular animal, only G, IGG, and IGGl predominant antibodies are actually isolated, which are complement fixing and neutralizing antibodies respectively. The solution is also analyzed for sodium, potassium, chloride, and bicarbonate. An HIV test is also performed on the serum to make sure that neutralizing antibodies are still present after the process. Once the antibodies are isolated out and all cultures are negative, and the immunoglobulin electrophoresis shows the presence of immunoglobulin in the IGG form, IGG and IGGl, the material, the antibody, is then quick frozen again and is now waiting to be used, either intradermally or intravenously.

Anti-HIV titer to qualify the goat donor.

One million particles of serotype HIV in one ml were used to immunize the goat every week for four weeks, and then it was repeated at six weeks. The immunogenic or immunological response is then measured by the presence of neutralizing antibodies. Once we get to a point where a dilution of 1-20 produces no cell fusion, then this goat is now used as the animal for us to be able to draw blood from and separate out neutralizing antibodies to prevent cell fusion and to treat patients. So, the goal is a dilution to 1 -20 as far as antibodies using serum to prevent cell fusion.

Biologic agents

Numerous references that talk about the selective infection of human CD4 cells by the simian and HIV immunodeficiency virus and productive infections associated with envelope glucoprotein-induced fusion. I cite for you the procedure for Proc Natl Academy of Science U.S.A. 86: 2443-2447,1989: The cells were grown in NPMI-1640 media (RPMI) supplemented with 10% fetobovine serum, glutamine, and gentamicin and were used for preparation of stock virus and virus neutralization assay. Cell cultures (9 milliliters) were inoculated with 1 ml of virus (Ten to the fourth TCID 50/ milliliters} and examined for cell fusion. When approximately 50% of the cells had fused, the culture was expanded by the addition of fresh cells. Cultures were further monitored for infectivity by fusion and reverse transcriptase. Supernatant fluid, approximately 240 mis, was collected and clarified by centrifugation. The stock contained ten to the fourth TCID 50/ml and CEMX174 cells. The virus was propelled at 27,000 rpms in an SW28 rotator, Beckman Instruments, Inc., Fullerton, California, for two hours at 4 degrees C, re-suspended in 2 mis of NET buffer 50 mM HCl, 5 mM ethylene biamine ester acetic acid, 10 mM TRIS hydrochloride, pH 7.4, and purified on a Sepharose CL-4D column (that is Pharmacia Diagnostic, Inc., Fairfieid, New Jersey).

End process control and specifications.

Sterile technique is maintained. The serum is still frozen at a -70 degrees, and then before it was even frozen, it was heat killed at 60 degrees for 30 minutes. Once the virus was injected and had fully been injected over the four- week span, and then re-injected at six weeks, there was no more virus present. So, the virus had already been injected into the animal. The process of separating out the serum from the blood was the usual sterile techniques which have been described was also used. Then, once the serum was separated, only - sterile containers and sterile beakers under a P2 hood were used with sterile pipetting. Once the precipitation reaction was over using ammonium sulfate, which by the way, ammonium sulfate, which is supersaturated in this manner, has been shown to be an extremely effective sterilization process because of the supersaturation of the ammonium sulfate to kill any living organism that may be present within the immunoglobulin itself. So, the ammonium sulfate even acts as a sterilization process. This particular process of separating out the immunoglobulins with the ammonium sulfate, followed by centrifugation, and then adding the buffer to the solution. Then, the dialysis was performed in a cold unit right at about 4 degrees C. This was done over a 24-hour span. This was continued until no ammonium sulfate was present. All of this is then still done under sterile technique. Once the dialysis process is completed, the solution is quick frozen in a -70 °C freezer, and kept that way until it is then cultured and analyzed for microbiological organisms, gram staining, etcetera, which has been described in the schemata. The lot number of this particular amount of immunoglobulin is maintained throughout the entire control specification process.

Storage

The antibodies are all frozen in freezers that have to be at least -20 to - 40 °C, which maintains the antibodies integrity. The storage can be from three years to maybe five years with the antibody maintaining its integrity. All of the product intermediates which were used are of no significance once the antibody has been isolated, purified, and frozen, and then noted to be ready for use in humans. Of note is that the usual microbiological process is for culturing, for gram staining, for acid fast staining, for fungal staining, for viral cultures, are all the usual customary measures that we use to verify sterility and contamination of any product, be it blood, be it cerebral spinal fluid, be it immunoglobulins that are going to be used in humans, be tested.

Clinical toxicology data

We do what is known as a test dose to see if the person develops an allergic reaction to the hyperimmune goat serum. We use 0.1 ml. Intradermally, wait approximately 30 minutes to see if we get an immediate reaction which is manifested as edema, erythema, and itching. If this reaction is negative, then the assumption is that an immediate sensitivity reaction is most likely not to occur. However, if it does occur, if this particular solution is given intravenously, advanced cardiac life support should be available, including any type of pressors, mass trousers, ventilator support, bronchodilators, and steroid therapy would be used in an appropriate manner. We feel that if a person does develop an allergic reaction and they are HIV positive, that the person should be pre-medicated with antihistamines and steroids in order to prevent or to minimize the allergic reaction. The allergic reaction does not preclude the person, however, from receiving a potential life-saving treatment because of a possible allergic reaction. By treating the allergic reaction and minimizing it, it is more to the patient's advantage than letting them proceed on with an active HIV infection that can progress to flank AIDS and then to death. So, to answer the toxicology question pre-clinically, all we can say is that we will use a test dose of 0.1 ml. Intradermally to see if an immediate allergic reaction occurs. We will then follow the patient over the next week to see if a delayed sensitivity reaction occurs, and that is going to occur within five days after injection of this particular antibody. If no allergy, rash, wheezing, or any of the routine allergic symptoms occur in this particular individual after one week, then it is assumed that this person does not have any hypersensitivity or allergy to the serum. That is our toxicology data.

Clinical protocol

The protocol itself is very simple, in that if we use an intradermal process, which means that the person is not as sick, we will use 0.1 ml. As a test dose for the first week to see if a person develops an immediate reaction within 30 minutes, or a delayed reaction after approximately seven days. This is for either the intradermal or the intravenous use. If we get no reaction, the assumption is made that the person has no hypersensitivity to the immunoglobulin. If we use the intradermal injection, we will elevate the dosage to 0.3 ml. Intradermal, and we will give that once a week for approximately four weeks. At the same time, we will monitor the person's viral load, their P24, their CD4 cell count, CD3 cell count, CD8 count, CBC with differential, their sedimentation rate, and obviously all of their clinical symptoms, including their weight, their appetite, and their sense of well-being. We will measure for kinins, which has been suggested by the immunologist. We will also measure the usual routine laboratory measurements such as the electrolytes, liver function tests, and kidney function tests to see if we are getting any destruction or adverse effects in these particular areas. The protocol in terms of an intravenous dose, we will start off with 0.1 ml. Per hour with this person in a hospital setting. Obviously, this person is going to be a much sicker person clinically, most likely bedridden, extremely cachectic, anorexic, with death very, very close. The only exclusion at this point in time would be that the person has some severe end-organ damage, but even at that the decision not to treat somebody would be very, very hard to make whether or not to treat someone. Under ideal conditions, we would rather for them not to have any end-organ damage, to make sure that they can tolerate the antibody from a system perspective. The intent would be to get the dosage up to at least 0.3 ml. Per hour for a 24-hour span, which is approximately 7 ml. In a day. We would continue this process under close observation for at least seven days. The rationale for this is that we know that a dilution of 1-20 over a seven-day incubation period invitro rendered complete elimination of cell fusion with the HIV virus with the human CD4 cells. So, this is our starting point. The amount of antibody actually needed may not even need to be that much, or it may need to be a little bit more. But, because we are in an unknown zone, some kind of data must be accumulated in order to tell us where to start and stop.

We are basically using a theoretical goal of 0.3 ml per hour for every day for seven days as our protocol. If we use it intradermally, we are talking about 0.3 ml intradermally once a week until the viral load is undetectable, the CD4 cells, CD8 and CD3 counts are all normal, white blood cell counts are normal, the patient is now gaining weight, appetite is back, and their overall sense of well being has improved as per the patient. Therefore, to increase the dosage past what we are recommending because it is a theoretical number and because we may be dealing with people who are extremely ill. If we do not get any adverse effects and the person is tolerating it, the decision to increase the dosage would be basically a clinical decision based upon how sick the patient is. We do not anticipate having to go above 0.3 ml per hour per day for seven days.

The active component presumably includes goat anti-HIV antibody and other antibodies forming the total antibody population. In some of the following passages we refer to Bio Immune Stimulating Protein, or BISP, as a convenient label for the active component. It seems to be the case that the active component in BISP is the goat anti-HIV antibody, along with other auxiliary or assisting molecules. Such assisting molecules may be proteins but might also be other kinds of molecules that play a key role in achieving the benefits of this invention. Summary of Drawings

Figure 1 to 8 give data for the treatment of patients in accordance with this invention.

Examples of the Invention

The present invention is now illustrated by the following Examples, which are illustrative but not binding on the present invention.

EXAMPLE I

PREPARATION OF CONCENTRATED SIMIAN

IMMUNODEFICIENCY VIRUS (SIVϊ

A macrophage-tropic strain of SIV (SIVmac239-17E) referred to as "SIV- 17E" was prepared by growing the virus in CEMxl74 cells. Sharma, et al., "Derivation of neurotropic lymphocytotropic parental virus: pathogenesis of infection in macaques," J Virol 66: 352-35556 (1992). The CEM l74 cell is an immortalised CD4-bearing human T/B hybrid cell line that is highly susceptible to SIV- induced cytopathicity (fusion) and permissive for replication by SIVmac. Hoxie, et al., "Biological characterisation of a simian immunodeficiency virus-like retrovirus (HTLV-IV): Evidence for CD4-associated molecules required for infection, "J Virol 62:2557-2568 (1988). Koenig, et al., "Selective infection of human CD4 + cells by simian immunodeficiency virus: productive infection associated with envelope glycoprotein induced fusion," Proc. Nat . Acad. Sci. USA 86:2443-2447 (1989).

The cells were grown in RPMI-1640 medium ("RPMF'l) supplemented with 10% fetal bovine serum, glutamine and gentamicin, and were used for preparation of stock virus and the virus neutralisation assay. Cell cultures (9 millilitre) were inoculated with 1 millilitre of virus (10⁴ TCID 50/millilitre) and examined for cell fusion. When approximately 50% of the cells had been used, the cultures were expanded by the addition of fresh cells. Cultures were further monitored for infectiviry by fusion and reverse transcriptase. Supernatant fluids (approximately 240 millilitres) were collected and clarified by centrifugation. The stock contained 10⁴ TCID 50/millilitre in CEMxl74 cells. Virus was pelleted at 27,000 rpm in a SW28 rotor (Beckman Instruments, Inc., Fullerton, CA) for two hours at 4 degrees centigrade, resuspended in two millilitres NET buffer (50 mM HCl, 5 nM ethylenediaminetetraacetic acid, 10 nM Tris hydrochloride, pH 7.4) and purified on a Sepharose CL-4B column (Pharmacia Diagnostics, Inc., Fairfield, NJ).

EXAMPLE II

NEUTRALISATION OF SIV IN VITRO Simian immunodeficiency virus (SIV) was prepared according to procedures given in Example I. Killed virus was used for the sake of safety. The immunogen is heat killed at 60 degrees for 30 minutes and then quick frozen in a -70 degrees freezer and stored and shipped in a frozen container. It is then heat killed once more at 60 degrees for 30 minutes.

Goats were raised by a veterinarian, and a candidate pregnant female animal was identified. This particular animal was isolated out from the herd itself. Being isolated out from the herd, this animal was initially examined and then shown to be physically normal and absent of any microbiological illnesses, including CAEV, for which the animal was tested to make sure that it is CAEV negative.

The pregnant female goat was exposed to the simian virus by intramuscular injection. The goat was injected with a one millilitre suspension of killed SIV at 1 x 10⁶ viral particles per millilitre once per week for three weeks. The goat's immunogenic response was augmented using the MPL® (RIBI IMMUNOCHEM RESEARCH, INC.) + TDM Adjuvant System (Sigma Chemical Co., St., Louis, MO) according to the manufacturer's instructions, i.e., administering intramuscularly 500 μl into each hind leg.

Following exposure of the goat to simian SIV virus, the animal's serum and/ or milk was obtained. Milk was obtained from the goat as soon as it became available, generally at about three weeks after the birth of the kid. The milk was frozen until subsequent use. To collect serum, blood samples of at least 10 ml were drawn from a large bore vein after the first week and weekly thereafter for twelve weeks. This regimen was selected to optimise the opportunity to first detect neutralising reagents. Each serum sample was obtained by centrifugation of the blood sample, frozen for transport to the laboratory, and subsequently tested for neutralising capability. The neutralising reagents were first detected on or about the eighth week. During subsequent weeks, samples were tested to monitor changes in neutralising reagent concentration. The amount of antibodies detected increased over time. Straight, untreated serum (without concentration) obtained from the blood sample taken at the twelfth week was used in the in vitro neutralisation studies.

A neutralisation assay was performed to demonstrate the ability of the neutralising reagents and cellular immunity in the goat serum to prevent infectivity of the SIV virus in vitro. SIV-17E virus (100 TCID So /millilitre) was incubated with doubling dilutions of the goat serum at 37° C for one hour. In a 96-well tissue culture plate 100 microliters of each SIV- 17E/ goat serum mixture was added to wells using three wells per dilution. Approximately 5 x 10⁴ CEMxl74 cells were then added to each well. The cultures were incubated at 37 degrees Centigrade and observed for fusion over a period of five days. Fusion was observed in control cultures within one day. The neutralising titer was taken as the highest dilution of serum which prevented cell fusion.

The results of the neutralisation assay are given in Table 1. Table 2 presents the neutralisation assay data as a percent of inhibition of SIV fusion sites by various dilutions of goat anti-SIV serum at day 2 post- infection.

A 1/20 dilution of the goat anti-SIV serum almost completely inhibited SIV infection of the CEMx 174 cells (97.2% inhibition). Dilutions of 1/40 and 1 /80 inhibited 92.4% and 83.7%, respectively.

Table 1

Average Number of Fusion Sites Over Time For

Various Dilutions of Goat Anti-SIV

Serum vs The Normal Saline Control

DIL NORMAL ANTI-SIV

1/20 70 2

1/40 72.5 5.5

1/80 75 13

1/ 160 80 34

1/320 85 58

1/640 85 85

There is a consistent drop in SIV fusion sites for the anti-SIV when compared with the normal saline control. There is a decrease in viral fusion sites as the concentration increases to a 1/20 dilution with about 100% of fusion inhibited, achieved after a 2 day incubation.

Table 2 Percent Inhibition of SIV Fusion Sites by Different Dilutions of Goat Anti-SIV Serum (Day 2 post-infection)

DILUTION

1 1/20 97.2

2 1/40 92.4

3 1/80 83.7

4 1/160 57.5

5 1/320 31.7

6 1/640 0

These results indicate that the goat anti-SIV serum contains potent neutralising reagents which can be used to block the infectivity of the SIV virus.

The similarity between HIV and SIV indicates that goat anti-HIV serum would be expected to block infectivity of HIV into human T cells.

EXAMPLE III

PREPARATION FOR HIV TREATMENT

Serum for the treatment of HIV was prepared in the following manner. A goat was inoculated with HIV- 13b virus, using an intra-muscular injection of HIV- 13b at a concentration of 10⁹ viral particles per ml. The virus was previously heat killed at 60°C for 30 minutes.

Blood samples were drawn after an appropriate interval, such as two weeks, for initial neutralisation studies as exemplified in Example II. In the optimised procedure, the goat is injected every week for four weeks, then at six weeks the animal is then bled to obtain the reagent.

Approximately 400 ml of blood is drawn from the goat under sterile technique. The area for needle extraction is shaved and prepared with betadine. An 18-gage needle is used to draw approximately 400 ml of blood from the animal. Of note is that the animal can tolerate approximately 400 ml of blood drawn without the animal suffering any untoward effects. The animal does not have to be sacrificed. The animal can then be re-bled in approximately 10 to 14 days after it replenishes its blood volume.

Fusion studies as exemplified in Example II were used to confirm the presence of neutralising antibodies reagents.

Once the presence of such reagents was confirmed blood was then taken from the goat at between 4-6 weeks, and centrifuged to separate the serum.

300ml of serum was then filtered to remove large clots and particulate matter. The serum was then treated with supersaturated ammonium sulfate (45% solution at room temperature, P3 hood) to precipitate antibodies and other material. The resulting solution was centrifuged at 5000 rprn for five minutes, after which the supernatant fluid was removed. The precipitated immunoglobulin was resuspended in phosphate-buffered saline ('PBS buffer', see Sambrook et. al. 'Molecular cloning, A Laboratory Manual', 1989) sufficient to re-dissolve the precipitate.

The solution was then dialysed through a membrane with a molecular weight cut off of 10,000 Daltons. Dialysis was carried out in PBS buffer, changed every four hours over a period of 24 hours. Dialysis was carried out at 4°C.

After 24 hours of dialysis the contents of the dialysis bag were emptied into a sterile beaker. The solution was diluted such that the mass per unit volume = 1 mg per ml. The dilution was carried out using PBS. The resulting solution was then filtered through a 0.2 micron filter into a sterile container.

After filtration, the solution was stored at -70°C for 24 hours.

Both the ammonium sulfate precipitation step, the filtration step and storage at -70°C result in the production of a sterile solution.

EXAMPLE IV

ADMINISTRATION OF REAGENT TO PATIENTS A test sample of 0.1 ml of the mixture prepared in Example III at a concentration of lOOmg/ml was administered subcutaneously to human patients. After 30 minutes the skin was examined for local reactions, including redness, and itchiness.

In the absence of any adverse response, 10 ml of the antibody solution at a concentration of 100 mg/ml in PBS was administered for three consecutive days subcutaneously, after which patients were tested at 30 days post injection.

Patient Response

Three patients were treated according to the above protocol. After injection, the total numbers of CD8 suppressive T cells, CD4 helper T cells and HIV-1 RNA was examined.

The following results were obtained:

Patient A 21 Jan 24 Mar

HIV 1 RNA 99365 2533

CD4 T cells 109 154

CD8 T cells 417 663

Patient B 10 Jan 22 Feb

HIV 1 RNA 75023 399

CD4 T cells 341 425

Patient C 11 Apr 28 Apr 17 May HIV 1 RNA 4688 4656 2111

CD4 T cells 537 1239 551

CD8 T cells 805 211 1 877

In all cases, HIV-1 RNA levels significantly decrease. Results for HIV RNA are given in a number of copies of HIV-1 RNA per 1ml of plasma (defined as viral load) . Changes of five fold or more in viral load are considered significant with regard to predicting disease progression and monitoring efficacy of antiviral therapy. In all cases the patient results indicate significant reductions in viral load.

Levels of CD4 helper T cells also rise in all individuals following the treatment with the extract.

EXAMPLE V

PATIENT RECORDS: ASSESSMENT AND EVALUATION

This example comprises a selection of data drawn from the records of patients who have undertaken a course of treatment using the reagent of the invention.

The protocol used was

TREATMENT REGIME. All medications are administered subcutaneously.

The treatment programme is divided into three stages: -

1. A primary stage designed to lower the viral load to undetectable levels and return the helper T Cells to within normal ranges.

2. A continuation stage designed to ensure the elimination of the virus.

3. A maintenance stage designed to monitor progress and re boost the immune system.

Each stage is further sub divided by clinical condition and assessed level.

NOTE

1. It is quite normal for labs to show an increase in viral load during the first seven weeks of treatment, since this is a measure of both live and dead virus, yet to be excreted.

2. In the event that an increase in viral load is noted during the first seven weeks, it has been our experience that Helper T cells show a major improvement at the same time and within a further two weeks the viral load will drop dramatically.

LABS TO BE DONE PRIOR TO FIRST TREATMENT AND AT SIX WEEKS PRIMARY STAGE CHILDREN 5 TO 13 YEARS

LEVEL 5 AND ABOVE

Children of clinical level five and above who are under 13 years of age receive: -

Day One A loading dose of nought point eight (0.8) ml and no further treatment for the next seven days

Day Eight A further nought point eight (0.8) ml

Day Thirty-Six A further nought point six (0.6) ml

Day Forty -Three A further nought point six (0.6) ml.

After day forty three Treatment should continue every thirty days, with a dosage of nought point four 0.4 ml until the viral load becomes immeasurable, an unexpected detrimental reaction occurs or the medical condition of the patient suggests that the dosage be changed.

PRIMARY STAGE CHILDREN LEVEL 4 AND BELOW

Children of clinical level five and above who are under 13 years of age receive: Day One A loading dose of one (1.0) ml and no further treatment for the next seven days

Day Eight A further nought one (1.0) ml

Day Thirty-Six A further nought point eight (0.8) ml

Day Forty- Three A further nought point eight (0.8) ml.

After day forty-three Treatments should continue every thirty days, with a dosage of 0.5 ml until the viral load becomes immeasurable, an unexpected detrimental reaction occurs or the medical condition of the patient suggests that the dosage be changed.

Depending upon the patient's tolerance, viral load and T cell counts, the dosage may be increased by 0.2 ml per treatment at the discretion of the Doctor.

Treatment should continue until the viral load becomes immeasurable, an unexpected detrimental reaction occurs or the medical condition of the patient suggests that the dosage be changed.

PRIMARY STAGE ADULTS LEVELS 5 & 6.

Adults and persons over 13 years of age of clinical level 5 or 6 receive a loading dose of two point five (2.50) ml and no further treatment for the next seven days.

A further two (2.0) ml is administered after 7 days with additional treatments of one point five (1.5) ml taking place at 3 week intervals thereafter.

Treatment should continue until the viral load becomes immeasurable, an unexpected detrimental reaction occurs or the medical condition of the patient suggests that the dosage be changed.

Depending upon the patient's tolerance, viral load and T cell counts, the dosage may be increased by 0.3 ml per treatment after the initial loading at the discretion of the treating Doctor.

PRIMARY STAGE ADULTS LEVEL 4 AND BELOW

Adults and persons over 13 years of age of clinical level 4 or lower receive a loading dose of 2.00 ml and no further treatment for the next seven days.

A further one point eight (1.8) ml is administered after the 7 days with additional treatments of one point two (1.3) ml taking place at 3-week intervals.

Treatment should continue until the viral load becomes immeasurable, an unexpected detrimental reaction occurs or the medical condition of the patient suggests that the dosage be changed.

Depending upon the patient's tolerance, viral load and T cell counts, the dosage may be increased by 0.2 ml per treatment at the discretion of the treating Doctor.

CONTINUATION STAGE CHILDREN AND ADULTS.

The continuation stage of the treatment commences when the patient's clinical condition and independent laboratory reports indicate that viral loads have become immeasurable and T cell counts are within the normal expected range. The full amount of medication administered is added cumulatively and half of the same amount is scheduled again for administration over the same or a shorter time period, depending upon the general clinical condition of the patient.

For example, if in the primary stage a person of 12 years old received 10ml of medication over a seven-week period, to obtain a viral load of zero they would in the continuation stage receive a further 5ml over the next seven-week period.

MAINTENANCE STAGE CHILDREN AND ADULTS.

After final treatment an following an initial six monthly check children are to be given a booster 0.4 ml injection and adults 2.0ml, with this being reviewed 12 months later.

In all cases, every six weeks a viral load, a CD4, CD3, CD8 Helper T cell count should be obtained and results placed on the clinical flow sheet.

NOTES.

1. When an individual has received their total dosage and the viral load remains a theoretictical zero for six months then the booster shot envisaged in stage 3 would be given.

2. An examination for the presence of the AIDS virus should in all cases be undertaken every two months until six months has expired and thereafter at twelve month intervals. If after 18 months no viral particles are present, one can only assume that the HIV/AIDS infection has been arrested.

3. Whilst in all instances T cell counts have substantially increased, occasionally during the first seven weeks of treatment some individual viral loads have risen, primarily because the measurement encapsulates both live and killed virus present in the blood stream including that which has been released from within sealed infected cells.

In each case the treating physicians notes have been first reviewed by the inventor and a joint detailed review has taken place.

In all cases independent laboratories have been used in order to determine variations in viral load and T cell counts.

Some treatments are incomplete or have been selected in order to give the reader a broad overview of the flexibility of the treatment, its application under clinical conditions and to promote constructive discussion as to its potential.

Treatments have been undertaken at a private licensed clinic in Mexico, involving patients, under proper informed consent, with all procedures based upon the United States FDA format.

Unfortunately, the costs of patient travel, with its attendant interruptions to a structured treatment protocol, were in some cases all too obvious. It is our belief, based upon direct clinical and laboratory observation, that when the reagent is approved for use within the United States or Europe, that even better results over a shorter time period will omlur.

The following records identify the active principal as Bio Immune Stimulating Protein, or BISP. This label does not represent any admission or denial that the active principal is a protein.

PATIENT:

Date of Birth: 29^th AUGUST 1963

TREA TMENT ASSESSMENT AND EVALUA TION

This Patient responded well to the Bio Immune Stimulating Protein (BISP), without any complications. It was noted that the AIDS complex, which consists of poor quality of life, decreased CD-4 and CD-8 cell counts, increased viral loads and P-24 values responded favourably, without complications.

This patient has in the past, tried multiple treatment regimens for his HIV/AIDS condition without good results. He was diagnosed with HIV twelve years ago and in the last five years has developed Karposi's Sarcoma, he also suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss and is currently clinically classified as being at level 6.

Ideally this patient should have received treatment on a more structured basis, however because of his inability to attend the treatment centre on a regular basis, he has received only two treatments (20ml total).

The Karposi's Sarcoma, has been eliminated and the viral load which peaked at 198,000 and bottomed at 900 dropped by a substantial log change of 2.3 almost four times greater than that of 0.5 logs which is currently considered clinically significant.

This patient during August 1999 underwent polyatomic oxygen treatment in Kenya with severe complications and is extremely pleased with the dramatic positive response that he has had to BISP he is committed to receiving this treatment under proper control clinical observation.

Of note when (BISP) was discontinued within one mouth his viral load began to increase, his CD-4 cell count dropped. When treatment resumed he immediately started to enjoy a major improvement in his quality of life a decrease in viral load, and an increase in the CD-4 and CD-8 cell count.

Meanwhile we in conjunction with his own physician, will continue to monitor and treat him as best we can.

This patient will continue to receive BISP medication until his viral load becomes undetectable and T Cell counts return to normal.

26^th October 2000

Refer to Figure 1

PATIENT:

Date of Birth : 20^th September 1975

TREA TMENT ASSESSMENT AND EVALUA TION

This patient was treated with the Bio Immune Stimulating Protein (BISP), without any complications.

She was diagnosed two years ago and suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, loss of weight and is currently clinically classified as being at level 3.

This patient for one year prior to treatment had tried multiple regimes for her HIV/AIDS condition with poor results. The complications and major side effects she experienced, included, diarrhoea, nausea, vomiting, and anaemia, ETC.

She has received three treatments, comprising a total of 20 ml of medication, which was dictated by her inability to attend the clinic on a more structured basis.

This patients viral load peaked at 75,023 and bottomed at 400 dropping by a substantial log change of 2.3 almost four times greater than a drop of 0.5 logs, which is currently considered clinically significant.

Also her CD-4 cell count increased from 340 to 440. This patients quality of life increased tenfold subjectively, physically, and she continues to do well. It is very important to realise that this person who was clinically at stage 3 of HIV/AIDS infection, is a good illustration that the sooner the treatment of BISP is administered the better the results subjectively and clinically.

During June / July 2000 this patient despite being made aware of the possible consequences became pregnant and we have therefore placed her treatment on hold.

It is intended to continue treatment following birth, meanwhile we in conjunction with her own physician will continue to monitor her.

26^th October 2000

Refer to Figure 2

PATIENT: AM 003

Date of Birth: 15^th January 1955

TREA TMENT ASSESSMENT AND EVALUA TION

This patient responded well to the Bio Immune Stimulating Protein (BISP), without any complications.

This patient has tried multiple treatment regimes for her HIV/AIDS condition with poor results. The complications and major side effects she has experienced, include: diarrhoea, nausea, vomiting, and anaemia, ETC. She has been blind in her left eye since 1996 due to retinal necrosis.

She was diagnosed with HIV ten years ago and suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss. When she started treatment she was classified as being at level 6.

Three treatments comprising a total of 20 ml were fitted around her ability to attend.

This patient will continue to receive medication until her viral load becomes undetectable and T Cell counts return to normal.

This viral load peaked at 56,000 and bottomed at 11 ,000 dropping by a substantial log change of 0.7. This difference of 0.5 logs is currently considered clinically significant. Also her CD-4 cell count increased from 390 to 480 after receiving BISP. This patient's quality of life increased tenfold subjectively, physically, and she continues to do well. Of note, her retinal necrosis has resolved. This implies that the retinal damage was most likely oedema.

It is very important to realise that this person who was clinically at stage six of HIV/AIDS infection, is a good illustration that the sooner the treatment of BISP is administered the better the results subjectively, and clinically.

Meanwhile in conjunction with her own physician, we will continue to monitor her.

26^th October 2000

Refer to Figure 3

PATIENT:

Date of Birth: 29^th September 1958

TREATMENT ASSESSMENT AND EVALUATION

This patient responded remarkably well to the Bio Immune Stimulating Protein (BISP), without any complications.

This patient has tried multiple treatment regimes for her HIV/AIDS condition without good results. The complications and major side effects she has experienced, include: diarrhoea, nausea, vomiting, and anaemia, ETC. She was treated at her own request with BISP.

She was diagnosed two plus years ago and suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss. When she started treatment she was classified as being at level 2.

Three treatments comprising a total of 20 ml were fitted around her ability to attend.

This patient will continue to receive BISP medication untilr her viral load becomes undetectable and T Ceil counts return to normal.

The patients viral load peaked at 75,000 and bottomed at 0.0 by a substantial log change of 4.9. This difference is ten times greater then the 0.5 log change, which is currently considered clinically significant. Also her CD-4 cell count increased from 500 to 560 after receiving BISP. This patient's quality of life increased tenfoid subjectively, physically, and she continues to do well.

It is very important to realise that this person who was clinically at stage two of HIV/AIDS infection has now sustained her condition in spite of the limited medication given.

This patient is also a good example that the sooner the treatment of BISP is administered the better the results subjectively, and clinically. This patient has been given another chance at life with minimal to or no side effects after receiving BISP.

Meanwhile in conjunction with her own physician, we will continue to monitor her.

26^th October 2000

Refer to Figure 4

PATIENT:

Date of Birth: 26^th JULY 1991

TREA TMENT ASSESSMENT AND EVALUA TION

This patient is a nine-year-old female who was born with HIV/AIDS. She has suffered numerous complications i.e. fever, chills, thrush, asthma, multiple episodes of pneumonia and has been absent from school numerous times since the illness began.

Her mother decided that the conventional treatment for HIV/AIDS was too toxic and causing her to suffer multiple side effects, which included diarrhoea, nausea, vomiting, fever and anaemia, etc.

This patient received BISP in the United States for seven consecutive weeks at a dosage of 0.3 ml per treatment, receiving in total 2.1 ml.

Prior to receiving the medication, she was being treated by the using the IL2 protocol and was experiencing numerous complications i.e. fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss. We do not know the effect of this protocol in this area.

This patient responded well to the Bio Immune Stimulating Protein (BISP), with a complete reversal of the HIV/AIDS complex.

She was completely compliant with the FDA protocol and it was noted that this patients viral load went form 186,000 and bottomed at 0 in seven weeks. A log difference of 5.3. This difference of 5.3 logs is consistent with an approximate tenfold decrease in the patients viral load and well exceeds the conventional clinically significant 0.5 log standard.

The patients CD-4 cell count went from 394 to 896; her CD-8 cell count went from 739 to 1210 after receiving seven treatments of BISP.

The first treatment was initiated in April 1998 and this response has continued until March of 2000 with the patient not receiving any BISP since June 1998.

Even though this patient has not received BISP in the last two years her quality of life has been maintained i.e. she has been able to maintain her attendance in school, no fatigue, no weight loss, and her appetite remains good.

It is very important to realise that this young person who was clinically at stage three of HIV/AIDS infection, was converted to a clinical level one without being allowed to join a proper protocol. She experienced a substantial improvement both subjectively, and clinically and has been given another chance at life with minimal to or no side effects.

This patient proves beyond a shadow of a doubt that when a protocol is adhered to even if it is based upon one designed for another treatment or if the medication is administered on sensible scheduled basis then positive results can be quickly achieved as is evidenced by the dramatic improvement in the quality of life given to this child and the obvious positive changes in her clinical data. With the introduction of a proper protocol that can be administered in structured manner it will be possible to predict the results with 100% accuracy.

Meanwhile we will continue to monitor her.

26^th October 2000 Refer to Figure 5

PATIENT:

Date of Birth: 01 MAY 1967

TREA TMENT ASSESSMENT AND EVALUA TION

This patient was diagnosed with HIV three years ago and has tried multiple treatment regimens with poor results. He also suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss and is currently clinically classified as being at level 3.

This Patient responded well to the Bio Immune Stimulating Protein (BISP), without any complications. It was noted that the AIDS complex, which consists of poor quality of life, decreased CD-4 and CD-8 cell counts, increased viral loads and P-24 values responded well.

Ideally this patient should have received treatment on a more structured basis and treatments were fitted around his ability to attend rather than adhering to a more correct clinical protocol.

The patient was monitored and reviewed despite being unable to finish the outlined clinical protocol and will continue to receive medication until his viral load becomes undetectable and T Cell counts return to normal.

The viral load peaked at 4,600 and bottomed at 420 after two treatments. This drop in the viral load equalled 1.1-log difference. This is a substantial log change by two times greater than that of 0.5 logs which is currently considered clinically significant. The patient received a total of 20 ml of BISP medication..

It is very important to realise that this young person who was clinically a stage three HIV/AIDS patient, has been given another chance at life with minimal or no side-effects.

Meanwhile we in conjunction with his own physician will continue to monitor and treat him as best we can.

26^th October 2000

Refer to Figure 6

PATIENT:

Date of Birth: 02 OCTOBER 65

TREA TMENT ASSESSMENT AND EVALUA TION

This patient who was diagnosed with HIV ten years ago is currently classified as being at clinical level 4, has participated in all current treatment protocols without satisfactory results. As a result of his HIV/AIDS condition he also suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes and weight loss.

This Patient responded well to the Bio Immune Stimulating Protein (BISP). It was noted that the AIDS complex, which consists of poor quality of life, decreased CD-4 and CD-8 cell counts, increased viral loads and P-24 values responded favourably.

This patient should have received treatment on a more structured basis and will continue to receive BISP medication until his viral load becomes undetectable and T Cell counts return to normal.

The viral load peaked at 99,000 and bottomed at 2,500 after two treatments. This drop in the viral load equalled 1.6-log difference. This is a substantial log change by three times greater than that of 0.5 logs which is currently considered clinically significant.

The patient received a total of 20 ml of BISP medication in combination with three retroviral drugs, these retrovirals did not cause the viral load to drop below 90,000 ccm until the combination treatment included BISP. . It is very important to realise that this young person who was clinically a stage four HIV/AIDS patient, has been given another chance at life with minimal or no side-effects.

Meanwhile we in conjunction with his own physician will continue to monitor him.

26^th October 2000

Refer to Figure 7

PATIENT: 8

Date of Birth: 14 ,t^uh^ι April 1968

TREA TMENT ASSESSMENT AND EVALUA TION

This Patient responded well to the Bio Immune Stimulating Protein (BISP), without any complications. It was noted that the AIDS complex, which consists of poor quality of life, decreased CD-4 and CD-8 cell counts, increased viral loads and P-24 values responded positively without complications.

This patient has in the past, tried multiple treatment regimens for his HIV/AIDS condition with poor results. He was diagnosed with HIV two years ago. As a result of his HIV/AIDS condition he also suffers from severe fatigue, weakness, night sweats, decreased appetite, enlarged lymph nodes, and weight loss and is currently clinically classified as being at level 6.

Ideally this patient should have received treatment on a more structured basis, however due to the "On Hold " status and the inability of the patient to visit the clinic on regular basis, three treatments were fitted around his ability to attend rather than a more correct clinical procedure. The patient was monitored and will continue to be reviewed despite his initially being unable to finish the desired clinical protocol.

The viral load peaked at 46,000 and bottomed at 2,000 in thirty day after four treatments. This drop in the viral load equalled 1.4-log difference. This is a substantial log change by three times greater than that of 0.5 logs which is currently considered clinically significant. The patient received a total of 20 ml of BISP medication .

It is very important to realise that this young person who was clinically a stage six HIV/AIDS patient, has been given another chance at life with minimal or no side-effects.

We have advised this patient to apply for an emergency compassionate wavier under the FDA rules and Federal United States Law in order that he is allowed to be treated properly in his home state, thus relieving him of the trauma of international travel and allowing him to be treated more consistently.

Meanwhile we in conjunction with his own physician will continue to monitor and treat him as best we can.

26^th October 2000

Refer to Figure 8

Claims

CLAIMS 1 A composition comprising antibody of a goat after challenge with an HIV virus. 2 A method of preventing HIV infection or treating an individual infected with HIV, comprising the steps of (1) exposure of goat immune system to HIV; (2) purification of antibody from the goat after HIV challenge; and (3) treatment of an individual with the antibody obtained in step 2 above. 3 Use of a composition comprising antibody of a goat after challenge with a human HIV virus in medicine. 4 Use of a composition comprising antibody of a goat after challenge with a human HIV virus in the preparation of a medicament of the treatment of HIV and AIDS. 5 A pharmaceutical composition comprising antibody of a goat after challenge with a human HIV virus, in combination with a pharmaceutically acceptable carrier. 6 A method of generation of a protective composition against HIV infection or replication, the method comprising the steps of:

(1) exposure of a goat immune system to HIV,

(2) purification of antibody from the goat after HIV challenge; and optionally,

(3) testing the antibody population for protective activity prior to use as a protective agent, through suitable in vitro or in vivo assays.

7 A composition, method or use according to any preceding claim, wherein the antibody is treated by one or more of the following steps: precipitation with 45% ammonium sulphate, freezing at -70°C for 24 hours or microfiltration.