ES2351207T3 - USE OF ANTI-HIV GOAT POLYCLONAL SERUM AS A THERAPEUTIC AGENT. - Google Patents

Abstract

The use of a goat anti-AHL class II polyclonal antibody in the preparation of a medicament for use in the treatment of a disease in a human being that involves a proliferative immune response, in which the disease is an inflammatory disease, an autoimmune disease, an axonal or nervous lesion, and in which the goat anti-AHL class II polyclonal antibody is induced after exposure to HIV.

Description

The present invention relates, in particular but not exclusively, to a therapeutic agent for the treatment of diseases that involve a proliferative immune response. BACKGROUND OF THE INVENTION

WO 97/02839 refers to Viral Suppression, Treatment and Prevention of Viral Infections. This provides a procedure to produce neutralizing antibodies for the treatment of a viral infection in a patient, which comprises the steps of:

to.

exposing a mammal to a virus, such that said mammal produces neutralizing antibodies against said virus and

b.

collect neutralizing antibodies from said mammal. In the examples, an HIV vaccine, called AAV2, is obtained by mixing HIV virus with HIV neutralizing antibodies obtained from a goat.

WO 01/60156 refers to Neutralizing Antibodies and Immunomodulatory Enhancement Compositions. This provides an immunomodulatory composition comprising:

heterologous antibodies specific for an antigen; Y

an antigen, in which the heterologous antibodies complex with the antigen by

combination with a pharmaceutical vehicle.

The examples are similar to those of WO 97/02839, and again, an HIV vaccine, called AAV2, is obtained by mixing HIV virus with HIV neutralizing antibodies obtained from a goat.

WO 96/20215 describes the use of class II anti-CMH antibodies, which interfere with the interaction between lipopolysaccharides (LPS) and its transducer molecule. It is said that this is beneficial in the treatment of various diseases and inflammatory disorders. The publication describes experimental results obtained from in vitro systems and in vivo treatment of mice injected with LPS and previously treated with class II anti-ALH mouse antibodies.

WO 98/13066 describes the use of class II anti-CMH antibodies to inhibit the function of T cells independent of T lymphocytes to prevent or reduce rejection of xenograft.

WO 02/07760 is related to a therapeutic agent. This provides an HIV infection prevention procedure or a treatment of an HIV-infected individual, which comprises the stages of

(one)

exposure of the goat immune system to HIV;

(2)

purification of the goat antibody after exposure to HIV; Y

(3)

treatment of an individual with the antibody obtained in step 2 above.

In preliminary clinical trials, based on the antibody product of WO 02/07760, patients with HIV have been successfully treated using a goat's serum after exposure to HIV.

Preferably, the treatment employs a serum composition that can be obtained by a procedure that involves inducing effective antibodies in a goat, extracting blood from the goat, demonstrating the neutralizing ability of HIV in the extracted blood, removing solids from the blood, precipitating the solids using ammonium sulfate or other suitable supersaturated precipitation agent, separate the precipitate, dissolve the precipitate in a suitable aqueous medium and dialyze the solution with a limit of 5 to 50,000 Daltons, preferably 7 to 30,000 Daltons, more preferably 8500 to 15,000 Daltons , especially, approximately 10,000 Daltons. The immunization procedure of the goat can be by intramuscular administration but other conventional techniques such as subcutaneous or intradermal administration can also be used. The purification process can also be completed by other commonly used fractionation action procedures (caprylic acid for example) provided that the total residue is used.

More particularly, the treatment typically employs a goat serum obtained in the following manner. Goat Serum Production Example

By intramuscular injection, lysed HIV-3b virus was inoculated into a goat, suspended in a normal commercial supernatant, using an intramuscular injection of HIV-3b at a concentration of 109 viral particles per ml. Previously, the virus was destroyed by heat at 60 ° C for 30 minutes. After an appropriate interval, such as two weeks, blood samples were taken for an initial evaluation. In the optimized procedure, the goat received a weekly injection for four weeks, six weeks later blood was taken from the animal to obtain the reagent.

Approximately 400 cc of blood was extracted from the goat by sterile techniques. The needle extraction area was shaved and prepared with betadine. An 18 gauge needle was used to extract approximately 400 cc of blood from the animal. It should be noted that the animal can tolerate a blood draw of approximately 400 cc without the animal suffering any adverse effects. The animal does not have to sacrifice. Afterwards, blood can be drawn from the animal in approximately 10 to 14 days, after it

Recover your blood volume.

The presence of possibly useful antibodies was confirmed. Then, once the presence of these reagents was confirmed, at 4-6 weeks blood was drawn from the goat and centrifuged to separate the serum. Then, to remove large clots and particulate matter, 300 ml of serum was filtered. The serum was then treated with supersaturated ammonium sulfate (45% solution at room temperature) for the precipitation of antibodies and other materials. The resulting solution was centrifuged at 5000 rpm for five minutes, after which, the supernatant liquid was removed. The precipitated immunoglobulin was resuspended in phosphate buffered saline (‘PBS buffer’, see Sambrook et al. ‘Molecular cloning, A Laboratory Manual’, 1989) enough to redisolve the precipitate.

The solution was then dialyzed through a membrane with a molecular weight limit of 10,000 Daltons. The dialysis was performed in PBS buffer, changed every four hours for a period of 24 hours. The dialysis was performed at 4 ° C.

After 24 hours of dialysis, the contents of the dialysis bag were emptied into a sterile beaker. The solution was adjusted so that the mass per unit volume = 10 mg per ml. Dilution was performed using PBS. Then, the resulting solution was filtered through a 0.2 micrometer filter in a sterile container. After filtration, the solution was divided into aliquots, into individual doses of 1 ml and stored at -22 ° C before use.

The reagent is therefore ready for use.

Changes can be made in this process, such as, for example, varying the concentration of ammonium sulfate or changing it to other reagents. Similarly, it is not necessary that the limit molecular weight of dialysis be 10,000 Daltons. THE INVENTION

As part of the treatments using goat serum, it was observed that there were other beneficial results.

The findings of a significant clinical improvement, in HIV-infected patients, as well as in patients with various conditions, such as multiple sclerosis and some cancers have the possible interconnection that all these diseases are in chronic inflammatory conditions.

In accordance with the present invention, the use of a class II goat anti-ALH polyclonal antibody is provided, as described in claim 1.

To the surprise of the inventors, they could easily demonstrate that goats immunized with HIV produce serum that can deactivate the mixed lymphocyte response assay, which is one of the classic activation assays used in vitro. Thinking that this could be some inherent property of goat serum, the inventors were impressed when the goat sera, which had not received injection, had no activity in these trials.

The inventors also demonstrated that this activity is closely associated with the activity against class II ALH antibodies. The inventors are unaware if this represents a molecular mimicry response or if the goat detects the class II ALH that the virus carries when it appears and detaches itself from infected cells, or that said ALH (CMH molecules) detaches itself separately from the virus but it is co-purified so that antibodies against said cell membrane components are also produced. In fact, it has not gone unnoticed by the inventors that in the preparation and induction of antibody responses that are modulating the beneficial therapeutic effect there may also be other cell membrane molecules, such as chemokine receptors and related molecules. However, if this is so, it seems that the anti-ALH II response is very dominant and the inventors think that it can play a significant, but not necessarily isolated, role with respect to the induction of the observed anti-inflammatory responses.

With the high titers of antibodies against ALH, both class II and class I in sera, those with the highest levels of antibodies are the ones that best deactivate mixed lymphocyte responses and appear to have the best efficacy in the clinical situation .

The inventors observed, with surprise, that antibodies against FAS, a major apoptosis ligand, were extremely high. In fact, they were correlated with CMH class II antibodies. Without wishing to be bound by any theory; the inventors hypothesized that antibodies with high titration against FAS could lead to immediate apoptosis a few minutes after the antibody adheres to the FAS antigen and the cells could be destroyed. This could lead to the inhibition of toxic chemokines, which are thought to be involved in MS disability.

The main use of the present invention is the use of a class II goat anti-ALH polyclonal antibody in the preparation of a medicament for use in the treatment of diseases with inadequately high levels of ALH. These are multiple sclerosis, rheumatoid arthritis, inflammatory diseases, autoimmune diseases and axonal or nerve injury.

The improvements observed in nerve function in people with traumatic nerve injuries suggest a property of neuronal growth factor and therefore it can be used for trauma, post-infectious lesions, for example, guillian-barre, malignancy lesions, etc. ., neuropathies associated with diabetes, alcoholism, poisoning with metals or other toxins, etc.

Using as immunogen a selection of antigens, preferably an antigen cocktail, suitable antibodies can be induced. It is possible that the use of a series of different antigens will give rise to antibodies that recognize common structures of the antigens providing a strong response in the patient. The inventors hypothesized that a selection of HIV isolates will provide epitopes with small variations in structure, and will result in a pan-antibody.

Therefore, to generate the serum, it is preferred to employ a cocktail of different HIV viruses, mainly produced in the CMSP, instead of using only T lymphocytes. The cocktail suitably contains 2, 3, 4, 5, 6 or more of these virus. Viruses are preferably in the form of lysates. Examples of preferred lysates include the following HIV-I isolates: 91US056, 92HT593, 92US723, 92US657, 92US660 and 92US714. Preferably the cocktail includes at least 1, 2, 3, 4, 5 or 6 of these particular isolates.

Activation of cells, for example with Concanavalin A, can offer advantages and, for example, higher levels of anti-dopamine activity can be achieved using Con

A. SHULA (not activated) did not have a significant increase in anti-R dopamine levels above baseline levels in D.O. However, HIV 3B (average of 12 goats and rabbits) does. There was also an increase in Dopamine R levels among CMSP cells activated with Con A in the cocktail.

Such antibodies can also be obtained using proteins containing peptides isolated from HIV lysates, synthetic peptides. Antibodies to lyse can be obtained and tested.

Being linked to the current theory of the inventors, it seems that the antibody of the present invention acts by suppressing cell proliferation of the type that needs HIV or other conditions that depend on said immune response. Therefore, the present invention finds application, for example, in the treatment of multiple sclerosis.

Knowing now the meaning of the anti-ALH II activity of the goat serum antibody, it is now possible to evaluate the probable usefulness of a series of said goat sera. A simple assay can assess the presence of anti-ALH II activity and allow the identification of a candidate serum appropriate for administration in patients.

Based on the ability of the serum to drastically inhibit RLM, it is suggested that this serum may act as a strong anti-inflammatory agent. There are several mechanisms by which this could be done and inhibition of ALH recognition is one of the most likely.

Another mechanism of action may well be due to the fact that the complement is involved in the activity of this goat plasma, since this is the only activity that is known to be prevented by heat treatment. The complement may well allow the weakened defector mechanisms of the patients to be active. For example; both the destruction of tumor cells and viruses to which antibodies are directed and the cell-mediated antibody-directed destruction require complement and there is a total absence of effect or mechanism in the absence of complement.

In general, injection into humans of antibodies derived from a non-human host is contraindicated. Normally a strong immune response is created against the same foreign antibodies. Surprisingly, however, it has been discovered that the use of goat serum extract does not cause the immune reactions that were anticipated with other foreign animal proteins. Immunosuppressed patients and normal individuals tolerate the injection of goat serum extract.

A destroyed virus is injected into a specifically identified goat, by intramuscular injection and allowed to incubate, then a measured amount of blood is drawn and modified conveniently.

The serum is optionally tested to determine the activity of the desired antibody and, optionally one or more anti-fusion ability, neutralization of the AIDS virus, its ability to enhance phagocytosis and its acceptability in the human body.

After inoculating the HIV virus to a selected goat, an immune response was detected after being exposed to a foreign protein antigen, according to previous studies. Then, the extracted serum was further modified to prepare it for use in humans.

Approximately two hundred people 10 years of age and older, of both sexes, have been treated satisfactorily with the medication, and none of them have shown relapses or suffered side effects.

The treatment is provided by a subcutaneous injection, in amounts that vary between 1/10 and 10 cc and is designed to deliver the medication as quickly as possible to the lymphatic system. Usually, the preferred dose for an HIV patient is 1 ml per week or as required, provided as a divided dose in both arms. Administration every 2 or 3 weeks is typical, then every 3 months. For patients suffering from cancer, 0.3 ml per week seems better.

In most cases, the treatment has been performed once every four weeks for a period of three months. The following are general comments on them:

one.

In less than sixty minutes, after the injection, a moderate to severe depression was reversed.

2.

Generally, within two hours after the injection, patients regained their appetite and actively sought food.

3.

Approximately two weeks after the first treatment, patients began to gain weight.

Four.

Independent laboratory reports confirmed that, from 4 to 6 weeks after the first treatment, viral loads and P24 values decreased substantially and that CD4 and CD8 cells increased dramatically.

5.

No side effects were observed.

It is important to keep in mind that, unlike current treatments, this medication does not require the patient to maintain a strict daily or daily regimen and depends on a simple injection that is administered weekly or monthly.

Preferably, the composition is purified and essentially only consists of a purified serum extract. In a further variation, the antibodies can also be purified in their entirety or selected and grouped according to a specific disease need from the complex mixture of serum or plasma by a conventional or any other suitable procedure, including, but not limited to , for example, immunoaffinity chromatography, saline precipitation, ion exchange chromatography, size chromatography, affinity chromatography, in combination as appropriate or desired.

Normally, a test dose is used to see if the person develops an allergic reaction against hyper-immune goat serum. After an intradermal injection, 30 minutes are expected to see if there is an intermediate reaction that manifests as edema, erythema and itching. If this reaction is negative, then it is assumed that an immediate sensitivity reaction is most likely to occur. However, an allergic reaction does not exclude the person from receiving a life-saving treatment due to a possible allergic reaction.

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

Unlike most other treatments, this product does not need the patient to maintain a strict daily or daily regimen to take the pill and depends on the administration of a simple periodic injection. The immune system of the patients who were treated more than two years ago and who had remained in the project, seems to have stabilized and returned to normal operating levels.

Unlike existing drugs, which patients often need to take daily for the rest of their lives, a typical treatment depends on a simple injection that a doctor will administer weekly or monthly. A normal treatment program is three months long, with a continuation procedure planned at six months, twelve months and two years or as needed if the virus reappears.

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

Preferably, the composition of the present invention is treated by one or all of the following: precipitation with 45% ammonium sulfate, freezing at -70 ° C for 24 hours or microfiltration.

The recovery observed in the majority of patients with MS, together with the high mood indicated at the time of receiving the treatment, has also prompted the inventors to investigate the activity against CNS receptors that may be involved in nerve stimulation and possible regeneration. The inventors have explored various sera to determine activity against a series of antigens and have observed activity against dopamine receptor, serotonin receptor, p75 nerve growth factor receptor and CXCL 10 chemokine (IP10).

Knowing now the meaning of the activity of the goat serum antibody against the dopamine receptor, the serotonin receptor, the p75 nerve growth factor receptor or the CXCL10 chemokine, it is now possible to evaluate the probable usefulness of a series of such sera. of goat. A simple trial can evaluate the presence of said activity and allow the identification of the candidate serum suitable for administration to patients. In particular, the combination of anti-FAS and / or anti-ALH antibodies may be important, together with antibodies against one or more of dopamine receptor, serotonin receptor, p75 nerve growth factor receptor or CXCL10 chemokine and therefore To guarantee its presence in the product, tests against various antibody activities could be conducted.

FIGURES

Figures 1 to 9 refer to the determination of the presence of anti-ALH antibodies of

class II;

Figures 10 to 16 refer to RLM studies;

Figures 7 to 19 refer to the determination of the presence of anti-FAS antibodies;

Figures 20-29 refer to the determination of the presence of antibodies

additional; Y

Figure 30 provides profiles for sera from immunized goats with different

immunogens EXAMPLES OF THE INVENTION

The following details represent the current process of the inventors for the preparation of the antibody product. Goat Serum Production Example

By intramuscular injection, a goat was infected with a viral lysate HIV cocktail formulated with Freund's adjuvant. Previously the virus was destroyed by heat at 60 ° C for 30 minutes. After an appropriate interval, blood samples, such as two weeks, were taken for an initial evaluation. In the optimized procedure, the goat received a weekly injection for four weeks, six weeks later blood was taken from the animal again to obtain the reagent.

Using sterile techniques, approximately 400 cc of blood was extracted from the goat. For needle extraction, the area was shaved and prepared with betadine. An 18 gauge needle was used to extract approximately 400 cc of blood from the animal. It should be noted that the animal can tolerate a blood draw of approximately 400 cc without the animal suffering any adverse effects. The animal does not have to sacrifice. Then, the animal's blood can be re-drawn, approximately in 10 to 14 days, after it recovers its blood volume.

The presence of possibly useful antibodies was confirmed, taking into account the activity of the desired antibody. Once the presence of these reagents was confirmed, blood was then taken from the goat at approximately 4-6 weeks.

Then, to create the reagent, the blood-based product was centrifuged to separate the serum. Next, 300 ml of serum was filtered to remove large clots and particulate matter. The serum was then treated with supersaturated ammonium sulfate (45% solution at room temperature), to precipitate antibodies and other material. The resulting solution was centrifuged at 5000 rpm for five minutes, after which the supernatant liquid was removed. The resulting immunoglobulin was re-suspended in sufficient solution.

phosphate buffered saline (‘PBS buffer’, see Sambrook et al. ‘Molecular cloning, A Laboratory Manual’, 1989) to re-dissolve the precipitate. The solution was then dialyzed through a membrane with a molecular weight limit of 10,000 Daltons. The dialysis was performed in PBS buffer, changed every four to 5 hours over a period of 24 hours. The dialysis was performed at 4 ° C.

After 24 hours of dialysis the contents of the dialysis bag were emptied into a sterile beaker. The solution was adjusted so that the mass per unit volume = 10 mg per ml. Dilution was performed using PBS. Then, the resulting solution was filtered through a 0.2 micrometer filter in a sterile container. After filtration, the

The solution was divided into aliquots, in individual doses of 1 ml and stored at -22 ° C before use.

In this way the reagent is ready for use. Plasma to Serum Conversion: Materials:

Reagent

Formula Quality Amount needed per liter of water

Sodium chloride

NaCl USP / EP / BP 8.76 g

Sodium di-acid orthophosphate

NaH2PO4 USP / EP / BP 0.2 g

Disodium acid orthophosphate

Na2HPO4 USP / EP / BP 1.55 g

Dextran sulfate

Sodium salt, molecular weight 500,000 100g

Water for irrigation

N / A Baxter 1 liter

1 x 150 ml sterilized Duran

N / A N / A N / A

Duran sterilized 2 x 2 liters

N / A N / A N / A

Magnetic stirrer plate and sterile magnetic flea

N / A N / A N / A

1 Sartolab or Midisart filter

N / A 0.2 µm N / A

6 Stedim bags of 1 l

1 sterile 100 ml bag

N / A N / A N / A

15 Preparation of 100 mg / ml dextran sulfate solution

In 1 liter of water, the necessary amount of sodium chloride, di- orthophosphate, is dissolved

sodium acid and di-sodium acid orthophosphate, to make 1 liter of saline

phosphate buffered (PBS).

20 When completely dissolved, 10 g of dextran sulfate are slowly sprayed

in 100 ml of PBS stirring magnetically. For complete solubilization, mix for at least 10 minutes. It is filtered at 0.2 µm in a sterile container (for example a 100 ml bag) and, if not needed for immediate use, stored at room temperature. This is the dextran sulfate stock solution.

5 Conversion of plasma to serum The volume of plasma needed to be treated is weighed using a conversion factor of 1.0275, ie 1000 ml weighs 1027.5 g. To each 1000 ml of plasma (final concentration of 1 mg / ml), 10 ml of the dextran sulfate stock solution is added. 10 Stir magnetically at room temperature for 30 minutes. It is transferred to 1 liter Stedim bags. It is centrifuged at 4000-400 rpm (relative centrifugal force = 4910 g) for a minimum of 30 minutes at 22 ° C. The supernatant is carefully aspirated into a 1 l Stedim bag and is

15 discard the precipitate. The necessary number of Stedim bags of 1 l is tared. The supernatant is filtered at 0.2 µm into 1 l Stedim bags. Each bag contains

approximately 500 ml of the filtered supernatant. The volume of serum is recorded in each bag. Each bag is numbered and identified.

20 If the serum is not going to be processed immediately it is kept cooled to 4-8 ° C for periods of up to 7 days or at -20 ° C for longer periods. Preparation of 36% Sodium Sulfate in w / v Materials:

Reagent

Formula Quality Amount needed per liter of water

Anhydrous sodium sulfate

USP / EP / BP 360 g

Water for irrigation

N / A Baxter 1.5 liters

1 x 2 liter sterilized

N / A N / A N / A

Magnetic stirrer plate and sterile magnetic flea

N / A N / A N / A

1 Sartolab or Midisart filter

N / A 0.2 µm N / A

2 bags Stedim 1 l

N / A N / A N / A

1.5 liters of water (1 ml = 1 gram) is weighed inside the sterilized Duran

of 2 l.

Water is heated to 30-35 ° C by placing the Duran in a pre-heated incubator during

at least 1 hour A sterile magnetic flea is inserted and placed in the magnetic stirrer. Slowly, while stirring, 360 g of sodium sulfate are added.

5 Stirring is maintained until the salt is completely dissolved and the solution is clear. If the solution is not hot enough or allowed to cool, the salt will begin to crystallize the solution. The salt can be re-solubilized by heating the solution at 50 ° C. 1 liter of the 36% w / v sodium sulfate solution is filtered at 0.2 µm in a 1 liter Stedim 10 bag. The bag is labeled conveniently. The remaining 500 ml is filtered into a different Stedim bag. 500 ml more are added

of water for injection by filtration and this bag is labeled as 18% w / v sodium sulfate. Precipitation of Serum Immunoglobulins using Sodium Sulfate. Observation. Before treatment with sodium sulfate, the plasma must be defibrinated by

15 the dextran sulfate process.

Materials:

Reagent

Formula Quality Quantity

Filtered serum

1 liter

36% sodium sulfate solution

N / A 1.5 liters

18% sodium sulfate solution

N / A 1 liter

Phosphate buffered saline solution

N / A 2 liters

Water for irrigation

N / A Baxter N / A

Depth filter

N / A N / A N / A

0.2 µm filter

N / A 0.2 µm N / A

Stedim diafiltration bag

N / A N / A N / A

The filtered serum in the Stedim bags is heated using the incubator set at 3020 35 ° C. 2 x 5 ml samples are taken. To the serum in the bags, an equivalent volume of the heated 36% sodium sulfate solution is added, for example 500 ml of saline solution: 500 ml of serum. Salt mixture: whey (white cream solution), mix carefully by shaking the bags manually or placing them on a tilting plate. Mix for 25 30 minutes. The bags are weighed and balanced and centrifuged at 4000-400 rpm for a

minimum of 30 minutes at 25-30 ° C.

The supernatant is carefully aspirated for disposal. It must be not to alter the precipitate. A sample of the 1 x 5 ml supernatant is taken.

To each bag of precipitate is added a sufficient volume of sodium sulfate at 18% w / v to make the weight of each bag approximately 1000 g.

Manually or by means of a tilting plate, it is mixed for a minimum of 10 minutes, to wash the albumin trapped in the immunoglobulin precipitate.

The bags are transferred to the centrifuge and the centrifugation is repeated as indicated above.

The supernatant is carefully aspirated for disposal. It must be not to alter the precipitate.

To each bag of precipitate, a sufficient volume of phosphate buffered saline is added, to make the weight of each bag approximately 1000

g. Manually or by means of a tilting plate, it is mixed for a minimum of 10 minutes and the bags are transferred to refrigeration to keep overnight. The bags are removed from conservation and it is verified that the precipitate has re

solubilized

It is filtered at 0.2 um into a diafiltration bag of the appropriate size.

Samples of 2 x 5 ml are taken. Bulk Formulation of the Immunoglobulin Solution

The ultrafiltration device (UF) is prepared with at least one 30,000 LPM membrane of 0.1 m2

The system is disinfected by recirculating a 0.5 M - 1 M solution of sodium hydroxide for a minimum of 30 minutes.

The sodium hydroxide solution system is drained and washed thoroughly with irrigation water until the pH of the water is neutral.

The diafiltration bag, which contains the immunoglobulin solution, is connected to the UF.

To the diafiltration bag, a bag containing PBS (10 x the volume of the immunoglobulin solution) is connected by a peristaltic pump.

The retained material line is connected to the diafiltration bag.

The diafiltered material line is connected to the residual bag.

The product is slowly processed through the ultrafiltration device, adjusting

the inlet and outlet pressures at 2.0 bar inlet and 0.5 bar outlet, using the pump speed and valve settings.

The immunoglobulin solution is concentrated at approximately 50 g / l. This is calculated taking into account the starting concentration and measuring the amount of liquid removed. For example:

The starting solution is 2 liters at 25 g / l = 50 g. The volume of solution removed in the diafiltrate is 1 liter. Therefore the concentration is 50/1 = 50 g / l.

The diafiltrate flow rate is measured and, to match this flow rate, the speed of the bag pump containing the PBS is adjusted.

Diafiltration continues until 10 volumes of buffer have been exchanged, that is, 1 liter of IgG solution requires 10 liters of PBS.

The concentrated diafiltered IgG solution system is drained into the diafiltration bag. The bag is disconnected and the PBS bag, which contains at least 1 liter of PBS, is connected directly to the system.

The PBS rinse solution is circulated again and this rinse is recovered inside the bag containing the IgG solution.

The bulk formulated IgG solution is filtered at 0.2 µm into a previously weighed Stedim bag.

When finished, a 1 ml sample of the filtered solution is taken and evaluated to determine the protein concentration.

The bag is weighed and the volume of the IgG solution is calculated.

Using the protein concentration and the volume of IgG, calculate the volume of PBS needed to dilute the solution to a final concentration of 10 mg / ml, for example:

IgG volume = 1300 ml. Protein concentration = 30 mg / ml

Total amount of IgG = 39,000 mg

Requirement = final concentration 10 mg / ml

Therefore, final volume required = 39,000 / 10 = 3900 ml. The current volume is 1300 ml. Therefore the volume of PBS that needs to be added is 3900 -1300 = 2600 ml.

To achieve the final necessary volume at 10 mg / ml., The PBS is filtered into the bag containing the bulk formulated IgG solution.

The reagent is ready for use and can be kept cooled for up to 1 week,

or frozen at -20ºC for higher conservation needs.

In these procedures changes can be made, such as, for example, varying the concentration of sodium sulfate or changing its reagents. Similarly, it is not necessary that the dialysis limit be 10,000 Daltons. Activity Mode Introduction

Confidential observations indicate that patients with AIDS-infected serum from goats immunized with HIV appear to improve, apparently drastically in some cases, and the inventors investigate a possible mechanism. Although HIV was injected into goats and the immune response is thought to be very specific, this basis does not explain the benefit observed for patients with multiple sclerosis. Scientific Bases of the Study

Although Dalgleish and colleagues had discovered, several years ago, the CD4 receptor as the HIV portal of entry, the classical interpretation that CD4 cells destroy HIV, which decreases significantly as AIDS infection progresses, was not an explanation. adequate pathogenesis. The main observations that do not fit well with this interpretation include:

(one)

The long period of time from infection to the onset of AIDS (approximately a decade)

(2)

The absence of disease in almost all chimpanzees and in approximately 5% of people infected with HIV. Chimpanzees have the same receptors and coreceptors as humans and the virus can be easily detected in infected chimpanzees whose CD4 count does not decrease.

Based on these observations, researchers have sought to define the differences between individuals who develop the disease after infection and those who do not. The main prognosis of the disease is the degree of immune activation after the initial infection and the level at which it persists. In other words, the greater the immune activation, the less time to develop the disease. A moderate degree of activation could lead to a longer time before developing AIDS and an absence of immune activation, even with virus replication, which is the norm in chimpanzees and rare in people.

It is important to indicate this, since the term "immune activation", in this context, means immune pan activation, that is, all aspects of the immune system are activated, which includes all subgroups of T lymphocytes as well as B cells. There are only three recognized causes of pan-immune activation.

The first of these is hyper-variability in the antigen that clearly presents HIV, however hyper-variability is detected in infected people and chimpanzees who do not progress.

The second possibility is a super antigen, which deflects the specific mechanism of antigens and causes activation of the entire immune system. Despite an initial enthusiasm a decade ago, no convincing super antigen associated with HIV has been observed to explain these findings. Westby and Dalgleish showed that the variability of the T lymphocyte repertoire, which had suggested the possibility of a super antigen, was exclusively as a result of the decrease in the population of CD4 and the differences could be explained by random destruction of CD4.

The only other explanation is that the body has detected a foreign cell or organ and created a chronic response against it. Clinically this occurs frequently after a transplant and in particular in bone marrow transplants and is known as chronic graft versus host disease (ICH). In this case, even the survival of some foreign cells is sufficient to activate the entire immune system leading to aggressive autoimmune responses similar to those observed in HIV-infected patients. The clinical characteristics of this disease are so extraordinarily similar to those of AIDS that before the discovery of the virus, a prominent immunologist from the United States, Gene Shearer of the NIH (United States), suggested that the disease could be induced by foreign cells transmitted by blood transfusions or sexual intercourse The discovery of the virus, within a few months and the transmission by Factor VIII (an acellular product) led to ignore the importance of this observation.

The absence of another explanation for pan-activation led Dalgleish and Habeshaw to seek evidence that the virus could mimic Class I or Class II ALH (which are the molecules that determine their own and present antigens). Although some GP120 sequences with lower homology with ALH had already been published, the inventors were able to identify a major region of the HIV envelope (GP120) that had marked structural homology with Class I and II ALH. Elizabeth Hounsell (MRC) was able to create a GP120 model based on the ALH molecules whose structure was known in detail. Based on these molecules and sequences, Habeshaw and Hounsell predicted that the virus could be detected as "strange" by people with ALH-B8 and also as "own" by people with ALH-B27. It is currently a matter of published facts that the only significant differences in ALH in a pan-HIV ALH marker study conducted in the UK by the MRC are that people with ALH-B8 develop the disease at a much faster rate than the rest of the population and that those who do not progress in the long term or who progress extremely slowly are ALH-B27.

Subsequent works have shown that:

(one)

Cytolytic T lymphocytes created in response to exposure to foreign cells (that is, different types of ALH) will also destroy HIV-infected own cells.

(2)

That the HIV viral envelope known as GP120 (which according to the inventors has structural homology with the ALH molecules that determine the same), can

bind peptides in a manner very similar to ALH. In addition, T lymphocytes induced against peptides and ALH that mimic GP120 will also respond to GP120 with the peptide but not to GP120 without the peptide suggesting that the ability to bind peptides may be very important in this confirmation and therefore the ability to produce chronic immune activation in susceptible subjects.

(3)

More recently the inventors have shown that the region in which peptides bind to ALH is present in GP120 and that, if this part of the molecule is removed, as Professor Sodroski kindly performed on Dana Faber in Boston, the peptides They will no longer join.

The impact of the previous observations suggests that the immune activation of the system could, in theory, be changed and therefore the disease may not progress. In this sense there are two main observations, one of which was a clinical study as a result of this science in which patients who could no longer take AZT, due to spinal insufficiency, took steroids, a very impressive classic anti-inflammatory agent that It cannot be taken for long periods without significant side effects. The inventors were able to observe a remarkable improvement of very sick patients, with classic characteristics very similar to ICH disease, on steroids and could demonstrate that the dose necessary to exert this effect was extremely high and not a practical proposal in the long term. More recently, a group from Duke University (United States) had shown that the viral load dropped in patients who were given steroids for HIV, which is currently being done more and more to combat what is considered autoimmunity (a classic characteristic of chronic graft versus host disease). In summary, there is a scientific basis that an inflammatory agent that acts particularly on the routes induced by class I and class II ALH could be beneficial in HIV infection. Essays and Results

To see if the goat sera had any of the inflammatory properties, the inventors added these to the reactions formed by mixing the cells of two different individuals with different genetic backgrounds. To the surprise of the inventors this was extremely effective in inhibiting this reaction. Using a large number of molecular antibodies, the inventors could see that the only antibody that was close to the goat's reactivity was a class II anti-ALH. Anti-class I antibodies only partially reduced this reaction. Of greater interest is that antibodies against the HIV V3 loop, considered by the majority of the scientific community as the primary target for a vaccine, actually make this pan-activation worse. This discovery could fit with the theory of immune activation, since the immune response against the virus actually provides growth and stimulation for the virus to "get out" and this could explain why vaccines targeting this region are so ineffective.

Taking into account that magical properties have often been attributed to goats and that the sera of other animals may have unexpected activity in specific tests, the inventors obtained sera from unvaccinated goats that had no activity. Then, the inventors additionally obtained serum from different goats with different vaccination programs. To the surprise of the inventors it was possible to demonstrate that these sera only detected HIV and ALH class II envelopes. In fact, smaller parts of the virus, which are known to have significant homology to ALH, are not detected by serum.

Therefore the inventors were extremely surprised to observe a main difference between the injection of the cocktail and the injection of an isolate, since the repertoire of HLA regions conserved in GP120 is actively recognized by the serum of the goat to which Inject the cocktail.

The interpretation of these data is summarized by the fact that goats that are injected with HIV produce a strong class II anti-CMH response that can be extended using different isolates. The following is the possible interpretation of how this happens:

(one)

The HIV envelope is so similar to class II ALH that it is recognized by the goat immune system that has a completely different repertoire of ALH.

(2)

The second possibility is that in the immunogen, that is to say the viral preparation, the ALH that is located on the surface of the cells through which the virus sprouts is being collected and that the immune response is not against the virus but against the virus. ALH co-associate.

(3)

The third explanation implies a combination of the two previous explanations. It is possible that the virus that sprouts has been fused with ALH as prepared and that this combination is strongly detected by goat sera. A recent publication demonstrates that, unless the virus sprouts through a cell with ALH and incorporates ALH into the membrane, the virus remains non-infectious. This means that there must be some ALH derived from the host cell to activate the mechanism of entry into the cell.

In this document, the inventors have shown that goats in which HIV preparations are injected produce a strong antibody response against the class II ALH molecule. Several diseases are known to cause destructive inflammatory lesions simply because the cells involved overexpress class II ALH. Although many types of autoimmune diseases share this property, one of the best known examples is that of multiple sclerosis. Therefore, it may happen that the immune response of the goat breaks the tolerance to these molecules and that this procedure may allow a strong anti-inflammatory process to occur in vivo that could be associated with a clinical benefit. There will be obvious advantages if this occurs during long-term high-dose steroid use when chronic administration of goat serum has no significant side effects.

The anti-HIV response, which is strongly class II anti-AHL, may well provide a vital connection as to the true need for an anti-AIDS vaccine and the intention of the inventors is to analyze this response to produce a candidate vaccine against The hiv. Goat sera immunized with HIV-1 and anti-inflammatory properties. Introduction

Goat sera were provided from animals immunized with HIV-3B viral lysate or an NIH viral cocktail of 6 different HIV-1 isolates. Some of the sera have been previously used as part of experimental treatment regimens with good results, however the mechanism by which they act is unknown. To determine a mechanism of action, the inventors explored serum in ELISA plates against HIV-1 gp120, ALH-DR1 and selected peptides from different regions of the virus surface glycoprotein, some carrying sequence homology with respect to ALH Materials and Procedures Serum and antibodies

Samples of goat sera immunized with the original HIV-3B that had been used for the treatment of patients were provided, together with samples of several goats immunized with HIV-3B viral lysate (animals No. 0125, No. 0126, No. 0127, No. 0128, No. 0129 immunized on September 14, 1999, September 21, 1999 and again on September 29 and November 7, 2000) as well as information related to blood collection and immunization data. For the ELISA, the inventors simply chose to explore samples of sera taken from production extractions in the last stages (November 30, 2000 and December 1, 2003). Animal No. 378 was immunized with a NIH viral cocktail consisting of the following HIV isolates (92HT593, 92US657, 92US660, 92US714, 92US723, 91US056). Immunizations were performed on November 7 and 28. Sera taken from a production extraction on January 25, 2001, were used for ELISA screening. Goat sera from Control were also provided. Along with the sera, the inventors included anti-human ALH-DR and anti-human ALH-DR, DP and DQ

(Pharmingen) both in the ELISA study and in mixed lymphocyte reaction studies and Mouse anti-gp 120 IgG (EVA3047) specific for the V3 domain of HIV-i-IIJb (IRIQRGPGR) obtained from Dr. J Laman through Dr Harvey Holmes (MRC AIDS reagents program, National Institute for Biological Standards and Control, Potter’s Bar, UK) during the study of RLM Peptides and Proteins

Recombinant HIV-i IIIB gp120 was produced in Chinese hamster ovary cells and provided by Dr. J Raina by Dr. Harvey Holmes in the MRC AIDS reagent program. Recombinant ALH-DR1 was the same protein provided by Prof. Don C. Wiley used during peptide binding experiments. Table 1 shows the HIV-1 peptides used in the study. Peptides ARP7022, ARP7 10, ARP740-23, -28, 42, -44, -45, -46 and -47 were obtained from Dr Harvey Holmes of the MRC AIDS reagent program. The P12 control peptide represents a disorganized sequence of the CS region of the gp120 of HIV-1 and was obtained from Prof. E. F. Hoursell, School of Biological and Chemical Sciences, Birkbeck College London, UK. Peptides were stored in aliquots at -20 ° C until use. ELISA

ELISAs were performed following a protocol similar to that described by Brown et al. (J. Immunological methods 1997, 200: 79-88). Peptides and proteins were mixed with 0.05 M Carbonate / Bicarbonate binding buffer pH 9.6 at 16 µg / ml and 1 µg / ml respectively and applied in duplicate on Immulon 4 LIBX (Dynex) high binding microtiter plates Technologies, INC. 14340 Sullyfield Circle, Chantilly, VA 20151-1683, USA) overnight at 40 ° C. The wells were blocked using 5 mg / ml casein in PBS and left overnight at 40 ° C. The goat sera were diluted 1/500 and 1/1000 times in PBS / 0.25% Casein while the purified antibodies were diluted 1/1000 and 1/3000 times respectively and incubated for 1 h at 37 ° C. The wells were washed 3 times with 0.05% PBS / Tween 20 using a Wellwash 4 (Denley) apparatus.

Immobilized goat antibodies were detected using peroxidase-conjugated anti-Goat / Sheep mouse monoclonal IgG, clone GT-34 (Sigma) diluted 1/1000 in PBS / 0.25% Casein / 0.01% Tween 20 and anti-ALH antibodies detected using a peroxidase conjugate with goat anti-mouse IgG (Sigma) incubated for 1 h at 37 ° C. After another round of washes, freshly prepared Ofhenylenediamine dihydrochloride substrate (OPD; SIGMA) was incubated in the wells, at room temperature in the dark and the reactions stopped at 15 minutes, adding 50 µl / well of H2SO4 2, 5 M. OD values were measured at 492 nm using a Microplate Reader.

Table 1. Peptides Used During Tests

ARP7022: (DQQLLGIWGCSGKLICTTAVPWNC) 24 residue peptide from a conserved region of gp41 of VHI (593-616) recognized by the most European and African HIV positive sera. Control peptide

ARP710: (VKIEPLGVAPTKAKRRVVQREKR) 23-residue peptide derived from the conserved C-terminal (CS) domain of HIV gp120 (486508) leading to the cleavage site of gp120 / 41. Contains structural homology with the ALH

ARP740 / 23: (RPWSTQLLLNGSLAEEEVV) 20 residue peptide derived from the C2 region of gp120 (252-271). It contains homology of sequence with the β chain of ALH DR4.

ARP740 / 28: (NTRKRIRIQRGPGRAFVTIG) (302-321) 20-residue peptide derived from the loop HIV gp120 V3

ARP740 / 42: (GQIRCSSNITGLLLTRDGGNS) (438-458) Contains homology with the β1 chain from DR.

ARP740 / 44: (NNESEIFRLGGGDMRDNWRS) (459-478) Contains sequence homology with the ALH-A2

ARP740 / 45: (GQDMRDMWRSELYKYKWKI) (469-488) Sequence recognized by M38, an antibody that reacts cross-linked with the ALHC and C5 region.

ARP740 / 46: (ELYKYKWKIEPLGVAPTKA) (469-478)

20 residue peptide derived from the C-5 end of gp120. It contains the first 3 remains of homology at end C.

ARP740 / 47: (EPLGVAPTKAKRRVVQREKR) (479-498) 20 residue peptide derived from the C5 region of gp120. Contains structural homology with

the ALH.

P12. (RAKTVERKVERRK) Disorganized sequence of the CS domain of HIV gp120 provided by E. Hounsell. Do not recognized by positive WV sera. Control sequence

Mixed Lymphocyte Reaction Inhibition Assays

Blood donors

Non-corresponding blood samples were provided with Class II ALH, with donor consent, through Liz Buckland of the South Thames Blood Transfusion Service, Tooting, London. CMSP Preparation

Freshly extracted venous blood was diluted in Hanks Balanced Saline Solution (HBSS; SIGMA), carefully applied onto a Histopaque (SIGMA) and centrifuged at 800 g for 25 minutes at 20 ° C. CMSPs were collected from the density interface with a Pasteur pipette, washed 3 times in HBSS and counted using a Beckman Coulter counter. Mixed Lymphocyte Inhibition Assays

For a given RLM, the CMSPs of two individuals incompatible with class II ALH were resuspended in RPMI 1640 medium, containing 10% thermo-inactivated human AB serum (SIGMA), 4 nM L-glutamine, Penicillin (1000 U / ml) and Streptomycin (100 µg / ml) (SIGMA) and stimulating or responding cells were designed. The cells were plated in triplicate with stimulators at 1 x cells / well and responders at 1 x 10 ∼ cells / well to provide a 10: 1 responder-stimulator ratio. To determine any inhibitory effect on cell proliferation exerted by goat serum and various antibodies, 3 µl of undiluted goat serum or 1 µl of purified antibody was added to the wells containing mixed cell cultures and incubated at 37 ° C in 5% CO2 for 6 days. The cultures were subjected to pulses of 1 µCi of tritiated methyl thymidine (3H-Thd; Amerhsam) on day 5, 18 hours before cell collection. Cells were collected using a Tomtec Harvester 96 Mach III cell collector on fiberglass filter meshes and the incorporation of 3H-Thd was measured using a Wallac 1450 microbeta liquid scintillation counter. The results are shown as average counts per minute (rpm). Results

Class II anti-ALH antibodies present in goat sera

The inventors investigated the extent to which different goat sera could recognize HIV gp120 peptides taken from different regions through HIV-1 gp120, many carrying sequence and structural homology with ALH (Table 1). In these trials, the inventors also included soluble HIV-1 gp120 and ALH-DR1. The results are shown in Figures 1 to 9. Nf represents the background levels. As a positive result, an OD level greater than 0.1 was taken given the absence of reactivity against any of the antigens observed with the preimmunized serum.

The different sera provided various results but generally directed towards a tendency to react with ALH-DR1. Pre-immunized sera did not react with any of the peptides or proteins screened and sera no. 0125 and no. 0126 also showed no great reactivity. However, the other sera demonstrated high levels of reactivity against ALH-DR1, in particular No. 0127 and No. 0128 that had a very high reactivity. To clarify, Figure 9 compares the reactivity against ALH-DR1 with AHL-DR anti-human and ALH DR, DQ and DP anti-human.

The anti-ALH activity is interesting for the reason that it most likely represents activity against the ALH molecules of the same virus cells that were cultured before immunization. As HIV tends to drag more ALH molecules in its envelope compared to the gp120 molecules it expresses, particularly class II ALH molecules that positively regulate in cells, the anti-gp 120 and anti-ALH class II activities combined can be what represents a good protective response against HIV. This may be the case of certain sera, particularly No. 0127 and No. 0128, in which a strong "allo" -response in these goats was induced by immunization with viruses that carried high levels of Class II molecules on their surface.

Serum No. 0378 of the goat immunized with the Viral cocktail of NIH demonstrated the best cross-reactivity in all rounds with respect to multiple HIV-i peptides with anti-ALH DR1 levels approximately equal to those of goat serum anti-LILV-3B original, suggesting that this serum could act better together against HIV. This serum reacts very well with a series of HIV-1 peptides that carry homology to ALH. None of the sera reacted with the disorganized control sequence P12, demonstrating that the reactivity was specific for LIIV peptides. Serums No. 0127 and No. 0128 represent the best sera in terms of binding to HIV-i gp120, although they contain very high amounts of anti-ALH DR activity. RLM inhibition by goat serum

With the RLMs of Figures 10 and 11, the inventors used individual cells.

Incompatible random. Although this did not necessarily produce the greatest proliferation (at that time there were problems with the media), the trend seemed to demonstrate inhibition of proliferation during MRF by goat serum (serum immunized with original HIV-3B used in each case). Subsequently, the inventors began using incompatible ALH-class II cells (Figure 12) that produced better proliferation. Like the anti-ALH DR antibody, goat serum successfully inhibits cell proliferation and may appear to have anti-inflammatory activity.

Based on what the inventors have observed from the ELISA results, it is very likely that anti-ALH DR antibodies are responsible for the inhibitory effects. The inventors highlight the role of anti-ALH DR instead of that of anti-class II, since, as a whole, it seems that the wide variety of antibodies does not inhibit proliferation anywhere with an efficacy similar to anti-ALH DR alone. Why this could happen is a mystery, however it could be that ALH-DR represents the main class II antigen involved in inflammatory activity and antibodies against it could result in signaling within cells to inhibit replication. Therefore, it might appear that goat serum acts as an anti-inflammatory agent, suppressing cell proliferation. This could predict a positive result in the case of HIV1 infection in which virus-induced cell activation is essential for viral replication. Although on the surface this may seem counterproductive, the introduction of a mild immunosuppressive agent could act to inhibit viral replication and control the disease since HIV, unlike HTLV-1 or HTLV-2 related retroviruses, does not immortalize cells and causes cancer, so it is totally dependent on cell activation for proliferation.

So far, no known T-cell super antigen for HIV has been identified that leads to the point of how a virus, which seems relatively ill equipped to activate the cells it infects, can cause disease. The inventors are investigating the theory that virus-induced aloactivation is at the root, either associated with the ALH molecules associated with the virus or with the molecular mimicry observed between HIV and ALH. Goat serum possibly contributes to the patient's well-being simply by limiting this pan-activation. Observations have been made along similar lines in an article by Saifuddin M et al. (Clin. Exp. Immunology 2000, 221: 324-331), who demonstrated the importance of class II ALH molecules, in particular ALH-DR, in viral replication. They indicated that antibodies against class II ALH molecules inhibited virus expression in cells expressing class II and suggested the implication of the inducible CMH class II transactivator (CIITA) in potentiating HIV-1 transcription. Indeed, these effects could be observed in the case of goat sera. This compares with the anti-loop V3 antibody, which causes increased proliferation and therefore could be counterproductive in the overall scheme of things. The highly variable V3 loop region of the gp120 not only allows viral escape mutants, but also acts as a decoy for the immune system for additional activation rounds. Unfortunately, the concern with this region is ultimately beneficial for the virus and keeps the immune response away from more conserved regions, against whose activity it might be more useful.

If the anti-inflammatory effects are, in effect, the main mode of activity of the sera, this could be useful for other various conditions in which hyperactivity and cell proliferation are at the root of the symptoms of the disease, for example multiple sclerosis For future experiments it is important to repeat ELISAs in all sera to confirm the degree of anti-ALH activity and gp120. In this case, the inventors are very interested in serum No. 0378 of the animal immunized with the NIH viral cocktail since it reacts effectively with many of the HIV peptides that carry homology with ALH antigens as well as ALH-DR1. Additional Experiments that are not part of the invention

The following data come from experiments similar to those performed previously, in which the inventors identified anti-ALH antibodies in goat sera that have been used as a form of treatment for volunteers suffering from multiple sclerosis and AIDS, when conventional therapy already It was not adequate. The inventors' work involves investigating an anti-inflammatory mechanism of action, proposed for goat serum, in the recovery of these patients after treatment. This was previously proposed when anti-ALH antibodies were known. However, the inventors now add a function for the anti-FAS antibodies present in goat sera, given their ability to induce apoptosis in a matter of hours to the cells expressing the FAS receptor.

FAS / APO-1 (CD95) is markedly present at high amounts in active lymphocytes, signaling by which it can induce the cell to commit suicide after cross-linking with the FAS ligand or with an anti-FAS antibody. The function of these molecules is extremely important, not only in the cytotoxic immune response to destroy infected cells, but also in the negative regulation of the immune response after the antigen has disappeared to prevent the constant proliferation of cells that could be detrimental to the guest Therefore, this route is considered vital for maintaining a balanced immune response.

In the case of HIV infection, it is observed that the immune system is abnormally active providing a lot of space for viral replication since it requires activated cells for this process. In patients with multiple sclerosis, highly activated cells cross the blood brain barrier and are responsible for lesions in the myelin sheath, resulting in plaque formation and loss of neuronal function.

The anti-inflammatory mechanism mediated through the activity of anti-ALH (at least anti-ALH DR) antibodies present in goat serum, could be the mechanism behind the patient's recovery. The inventors have now discovered anti-FAS antibodies present in goat serum, to a greater or lesser extent, and propose to combine the functions of anti-ALH and anti-FAS antibodies in this proposed anti-inflammatory mechanism for the mode of activity both in patients with multiple sclerosis as in patients with AIDS. The inventors propose that these antibodies are targeting the most highly active cells resulting in their death while anti-ALH antibodies suppress any immune activity. This could be continued later by a drastic reduction in the production of cytokines and chemokines resulting in the termination of the immune attack on myelin in the case of multiple sclerosis or the reduction of viral load in the case of HIV-infected patients, given the absence of activated cells and the death of cells that secrete or are about to secrete viruses. Experimental Work

The inventors began looking for anti-FAS antibodies given observations that goat sera from responders damaged cells when added in mixed lymphocyte (RLM) reactions. MRLs are the result of lymphocytes from 2 different people with different types of ALH that react in a non-specific manner, resulting in lymphocyte proliferation and further renewal. The inventors consider this to be a good system for testing anti-inflammatory antibodies since autoimmune reactions are aimed at inappropriate expression of auto-peptides in class II ALH molecules.

Figures 13 to 16 are photographs taken of some recent MRLs, with and without serum.

Figure 13 is a photograph of a mixed lymphocyte reaction after 72 hours.

Figure 14 is an RLM mixed with Pre-immunized serum after 72 hours.

In none of the cases were there significant changes with respect to what was observed with the RLM itself. In fact, pre-immune sera promote RLM.

Figure 15 is an RLM with serum No. 025 added. This serum comes from a goat that was considered insensitive to immunization in which a reduced amount was present.

or insignificant of ALH and / or FAS antibodies, something that correlates with the previous ELISA results when the inventors identified anti-ALH DR in responders.

Figure 16 is an RLM with serum No. 0127 added, which has proven to be a very good responder. There is significant cell destruction of many of the CMSPs in the RLM. This result is similar for all strong responders along with the serum preparation that is currently being used for treatment.

The inventors decided to look for an antibody capable of inducing the destruction of very active cells, such as an RLM. Therefore, the inventors sought the presence of an antibody against FAS that might be able to cross-react with the FAS receptor and induce the signals necessary for apoptosis to occur. Therefore, the inventors performed an ELISA against FAS that demonstrated the presence of said antibodies in the sera of goats that were considered the best responders and that complemented the previous results with anti-ALH antibodies.

These results, together with the previous data of the inventors, correlate well with what has been observed in the results of the inventors' RLM, with reduced cell proliferation due to cell destruction.

From recent ELISA results, the inventors carried out the identification of possible additional active components in goat sera that had been used as a form of treatment in volunteers suffering from multiple sclerosis (MS) and AIDS in which therapy Conventional was no longer adequate.

The results of an ELISA scan at a dilution of 1 in 1000 of the pre-immunized sera are indicated below. The antigens screened were Ovalbumin, ALHDR1 (DR1) Fas, the p75 nerve growth factor receptor (rFCN), the serotonin receptor (R Ser), the Dopamine receptor (R Dop), CXCL 10 and Interferon induced gamma monokine (MIG).

It is already evident that the antibody against the dopamine receptor is present at high concentrations in the sera while there is no activity against any of the other antigens. However, there is activity against many of these antigens in the original 3B serum, which had previously been used for treatment, and in serum 0127 produced from a goat immunized with HIV IIIb produced in a T-lymphocyte line, which includes activity against rFCN, the dopamine receptor and CXCL10.

The activity against ALH and FAS is again evident and is in accordance with the anti-inflammatory mechanism already proposed by the inventors. According to this topic, activity against CXCL10 and the nerve growth factor receptor p75 (R FCN p75) can now be added. The antiserum against CXCL10 has recently proved beneficial by greatly reducing the severity of the disease in the murine multiple sclerosis model, through the reduction of CD4 + T lymphocytes and CNS macrophage invasion, decreasing the expression of the TH1 IFN-γ cytokine and increasing remyelination. This is interesting since local cells in inflammatory lesions commonly produce CXCL 10 that binds to the walls, attracting inflammatory TH1 cells through the CXCR3 receptor. Therefore, the neutralization of this chemokine or the blockage of its receptor by antibodies in goat serum could well be part of the mechanism of anti-inflammatory action proposed by the inventors for goat serum.

On the other hand, antibodies against the low-affinity R FCN p75 nerve growth factor receptor are associated with cell death in HIV-infected monocytes and macrophages preventing FCN from reaching the receptor, which again represents another inflammatory mechanism. Interestingly, the inventors have discovered that the strongest levels of antibodies against these receptors are present in sera 0378 produced through the immunization of goats with a cocktail of 6 different HIV viruses produced mainly in CMSP instead of just T lymphocytes. The activity remains very high even with a dilution of 1 in 4000.

Therefore, the inventors added the activity against these antigens in accordance with the proposed anti-inflammatory mechanism of action for sera in the case of both diseases. The inventors also added activity against dopamine and serotonin receptors that are responsible for the feeling of well-being associated with the treatment.

The inventors explored many new goats (707-718) that had recently been vaccinated with HIV along with the pre-immune serum of these goats. Although none of them showed antibodies against the dopamine receptor, such as the original pre-immune sera shown above, some of these goats had, on average, a higher level of said antibodies compared to goats from Wales that had never been vaccinated against rabies. The higher levels observed in the original pre-immune sample could be due to exposure to some environmental factor at some time in the past followed by a strong response.

Claims (3)

1. The use of a class II goat anti-AHL polyclonal antibody in the preparation of a medicament for use in the treatment of a disease in a human being that involves 5 a proliferative immune response, in which the disease is an inflammatory disease, an autoimmune disease, an axonal or nervous lesion, and in which the goat anti-AHL class II polyclonal antibody is induced after exposure to HIV.

The use of claim 1, wherein the antibody has anti-ALH class II and anti-HIV activity.

3. The use of claim 1, wherein the disease is multiple sclerosis.

The use of claim 1, wherein the disease is rheumatoid arthritis.

5. The use of claim 1, wherein the disease is neuritis.