MX2008001923A - Intravenous immunoglobulin composition - Google Patents

Abstract

A method for preparing a concentrated, immunoglobulin composition for treating subjects vaccinated against or infected with a pathogenic microorganism, comprising:(a) selecting a population of individuals previously vaccinated against one or more antigens associated with the pathogenic microorganism;(b) determining the level of specific antibodies immunoreactive with the pathogenic microorganism in a blood or blood component of the individuals to identify very high titre individuals having a very high titre of the specific antibodies;(c) combining blood or blood components comprising immunoglobulins from the very high titre individuals;and (d) purifying and/or concentrating the product of step (c), thereby obtaining a concentrated immunoglobulin composition. Also disclosed is a concentrated immunoglobulin composition comprising specific antibodies immunoreactive with a pathogenic microorganism, characterized in that the titre of specific antibodies of the composition is at least 5 times higher than the average titre of specific antibodies of a population of individuals previously vaccinated against one or more antigens associated with the pathogenic microorganism. The composition has a relatively high protein concentration and a low percentage of protein aggregates, and is therefore suitable for both iv and im administration. In a preferred embodiment, the pathogenic microorganism is smallpox virus or vaccinia virus.

Description

COMPOSITION OF INTRAVENOUS IMMUNOGLOBULIN FIELD OF THE INVENTION The present invention relates to an intravenous immunoglobulin composition, a method for its preparation, the forms of the products that contain it and the therapeutic uses of said composition.

BACKGROUND OF THE INVENTION The smallpox vaccine contains the virus Vaccinia vivo, a virus that belongs to the family of orthopoxviruses and closely related to the variola virus, the agent that causes smallpox. Neutering the antibodies induced by Vaccinia against protects against other orthopoxviruses. Although the efficacy of Vaccinia was never demonstrated in controlled trials, epidemiological studies indicated a high level of protection against smallpox that lasts for 5 years or less after the first vaccination and a substantial, albeit diminished, immunity that may persist for more than 10 years. Antibody levels after re-vaccination may be longer, conferring a longer period of immunity than occurs after primary vaccination alone. For most people, the Vaccinia vaccine is safe and effective. In a non-immune person, which is not REF..190079 immunosuppressive, the expected response to primary vaccination is the development of a papule at the site of vaccination 2-5 days after the percutaneous administration of the Vaccinia vaccine. The papule becomes vesicular and then pustular and reaches its maximum size in 8-10 days. The papule dries and forms a scab, which separates after 14-21 days after vaccination, leaving a scar. Most people experience normal reactions, typically mild to the vaccine, such as swelling and tenderness in regional lymph nodes, fever, erythematous or urticarial rash, and inadvertent inoculation. Most of these cases are resolved without any treatment. Some people experience moderate or severe reactions, which require medical attention. Such complications are rare but occur at least 10 times more often in primary vaccinates than in re-vaccinated persons and more frequently in infants and immunocompromised subjects than in older children or adults. In the past between 14 and 52 subjects for every 1 million people vaccinated for the first time experienced reactions that could potentially endanger their lives. The following complications of Vaccinia vaccination that require medical attention are: Vaccination eczema: Serious rashes caused by the spread of the infection on the skin. It occurs in patients with a history of eczema or atopic dermatitis. The lesions appear at distant sites when the virus spreads through the body. The disease is usually mild and self-limiting, but it can be severe or fatal. The most serious cases among the recipients of the vaccine occur in primary vaccinates and are independent of the activity of the underlying eczema. Progressive vaccines (or Vaccinia necrosum): Continuous cutaneous infection with progressive tissue necrosis. The lesion may progress for several months and secondary lesions may develop elsewhere in the body. Infection is more common in primary vaccinates, immunocompromised subjects and in children and is frequently fatal. The strain of the vaccine virus influenced the occurrence of this complication, which was greater in Europe than in the United States. Generalized vaccinia: It is characterized by a widespread erythematous maculopapular rash, spread indiscriminately throughout the body. The papules become blisters and heal within 15 days. It is believed that the generalized Vaccinia results from the circulatory dissemination of the virus in normal individuals. The irregularity of the lesions and the healthy immune system of the affected patients differentiates this disease from the erythematous rash and accidental Vaccinia. Post-vaccinal encephalitis: Brain inflammation. It is a serious and rare complication and its relationship with the Vaccinia virus is still unknown. Encephalitis produced in children under 2 years of age is characterized by an incubation period of 6-10 days and is associated with degenerative changes in ganglion cells, perivascular hemorrhage and generalized hyperemia of the brain. The symptoms are the same associated with general encephalitis, including intracranial pressure, myelitis, seizures and muscle paralysis. A second form of the disease occurs in older children and adults. This is characterized by an incubation period of 11-15 days and is associated with symptoms of allergic response with destruction of the perivascular myaline coat. The strain of the vaccine virus influenced the occurrence of this complication, which was greater in Europe than in the United States. Two potentially fatal adverse effects that emerge from smallpox vaccination are myocarditis and ischemic cardiac events. Patients who experience heart disease after vaccination report shortness of breath, palpitations and chest pains. Death due to myocardial infarction is a marked possibility. Based on past experience, it is estimated that between 1 and 2 people of 1 million people vaccinated can die as a result of life-threatening reactions to the vaccine. In addition, subjects with weakened immune systems or certain skin conditions are susceptible to severe reactions, so they are excluded from vaccination with Vaccinia, unless you have been exposed to the virula virus. Intravenous immunoglobulin (IVIg) is a known medical product, consisting of a non-specific immunoglobulin solution obtained by the plasma combination of a plurality of individuals. Vaccinia immunoglobulin (VIG) is a solution of immune globulins obtained from a plasma concentrate of subjects who were vaccinated with the smallpox vaccine. The solution therefore contains a relatively high titre of anti-Vaccinia antibodies. The preparation of the commonly used VIG was an intramuscular (im) product (VIG-IM), produced by Baxter Healthcare Corporation in the 1990s. Because it contains a high proportion of added proteins, it was administered only intramuscularly and could not be used intravenously (iv). Plasma donors are not selected for their high levels of anti-Vaccinia antibodies, therefore the effectiveness of VIG treatment is limited and large volumes or multiple injections are necessary to obtain an effective level.

In general, an initial dose of 0.6 ml / kg. of body weight (about 100 mg / kg = 7 grams / adult treatment) was injected intramuscularly and the subsequent administration was determined according to the course of the disease. In severe complications of Vaccinia as in cases of eczema of vaccination and progressive Vaccinia were used up to 1-10 ml / kg. These high doses are divided into smaller units joined and injected intermuscularly in multiple sites, dispersed for a certain period of time. After the recent threat of bio-terrorist attacks, a great effort was devoted to producing VIG for intravenous administration (VIG-IV). There are available VIG-IV preparations (C-VIG, Cangene, Acambis / Baxter, Dyn port). No product is currently developed in the European Union. Treatment with VIG-IV requires medical attention and therefore must be administered within the framework of a medical center. The VIG was indicated for accidental implantation related to extensive lesions, vaccination eczema (EV), generalized Vaccinia (GV) and progressive vaccinia (PV). VIG is not recommended for mild cases of accidental implantation, limited or mild generalized vaccines, erythema multiforme or post-vaccine encephalitis. VIG may be beneficial in the treatment of ocular vaccinia that results from inadvertent implantation. When a Ocular vacuity with keratitis, the consideration of VIG should include the possible increased risk for corneal scarring. It was suggested that Vaccinia immune globulin is also valuable in prophylaxis after exposure to smallpox, as long as it is administered in the first week after exposure and concurrently with vaccination. Isolated vaccination is recommended for those who have contraindications to the vaccine, unless it has been more than a week after exposure. At that time, the administration of both products is recommended, if available. U.S. Patent No. 4,174,388 (McAleer et al) discloses hepatitis B immune globulin prepared from individuals who exhibit an increase in hepatitis B antibodies on the surface of at least 2,000 PHA units / ml. after immunization with hepatitis B surface antigen. U.S. Patent No. 4,617,379 (Dobkin et al) discloses the preparation of serum hyperimmune globulin against cytomegalovirus (CMV). Serum globulin is prepared from fresh normal plasma from donors who were not vaccinated with the CMV vaccine and who were screened for higher than normal CMV antibody titers. Plasma containing high titers of antibodies to CMV is collected and fractionated to obtain the product.

U.S. Patent No. 6,692,739 (Patti, et al) discloses a method and composition for passive immunization of patients infected or susceptible to infection by Staphylococcus bacteria. The composition is prepared from a plasma concentrate obtained by combining individual blood samples or blood components having higher than normal antibody titers; occurring naturally in an antigen of Staphylococcus bacteria. Samples of blood or blood components are obtained from a normal, unvaccinated population. In an alternative embodiment, the selected proteins or peptides are administered to a host to induce the expression of the desired antibodies, high titer serum or plasma concentrate is collected from the host and the concentrate is administered to a patient in need, optionally after its purification and concentration. WO 03/049117 discloses an injectable immune globulin intravenously with a high titer of antibodies to orthopoxvirus. The immune globulin is prepared by vaccinating the plurality of donors with an orthopoxvirus vaccine, isolating the plasma from each of the donors after a sufficient period of time to allow the production of antibodies against the orthopoxvirus vaccine and preparing an injectable immune globulin. of plasma. It also reveals the method to treat or prevent an orthopoxvirus infection and a method to treat or better the symptoms associated with the adverse reaction to orthopoxvirus vaccination.

BRIEF DESCRIPTION OF THE INVENTION In the present it was surprisingly discovered that a certain percentage of the vaccinated population possesses exceptionally high titres of antibodies and this high titre remains stable over time, after successive plasmapheres. This discovery was applied in the present invention to obtain a novel method and product. Thus, in one aspect, the present invention provides a method for preparing a concentrated immunoglobulin composition, which consists of: (a) selecting a population of previously vaccinated individuals against one or more antigens related to the pathogenic microorganism; (b) determining the level of the specific immunoreactive antibodies to the pathogenic microorganism in the blood or blood component of the individuals to be identified, individuals with a very high titer who possess a very high titer of the specific antibodies, (c) combine blood or blood components comprising immunoglobulins of very high titre individuals; and (d) purifying and / or concentrating the product of step (c), thereby obtaining a concentrated immunoglobulin composition.

In a preferred embodiment of the invention, the method also comprises the aliquot stage of the concentrated immunoglobulin composition in the form of a low volume unit dose. In one of the most preferred embodiments, the low volume dose consists of a vial or a pre-filled syringe. The use of a low volume significantly reduces the risk of side effects and facilitates the administration of the product. In the present specification terms will sometimes be used which will be understood according to the following definitions: Blood component - a fraction of whole blood comprising immunoglobulins. Plasma is a preferred blood component. Specific antibodies - antibodies that bind specifically to a particular antigen and not others. There may be counter-reactivity, however specific antibodies have a higher binding affinity, in a significant way, to the specific antigen. An example is the antibodies that bind to an antigen associated with the Vaccinia virus. Very high titer - high concentration of specific antibodies in an immunoglobulin solution, such as a blood component, generally at least 5 times, and preferably 10 times higher than the average concentration of the specific antibody in an immunoglobulin solution similar in the general population. A very high titer preferably refers to a titer exceeding 5 times and particularly 10 times greater than the average concentration of the specific antibody in a similar immunoglobulin solution obtained from individuals who were previously exposed to an antigen that causes the production of said specific antibody, like being through vaccination. A very high titre can also be determined by measuring the ratio of the specific antibody to the overall immunoglobulin content in the immunoglobulin solution, such as a ratio that is 5 times and preferably 10 times higher than the ratio in an immunoglobulin solution obtained from individuals who they were previously exposed to an antigen that causes the production of said specific antibody. In the context of the invention, individuals who were previously exposed to an antigen that causes the production of said specific antibodies are individuals typically vaccinated against a pathogenic microorganism, such as, for example, the Vaccinia virus. Very high-grade individuals - Individuals whose blood contains a very high specific antibody titer. Concentrated immunoglobulin composition - a composition comprising specific antibodies to a high titer. OmriUnits / ml - a measure of antibody titer arbitrary, calculated using the ELISA immunoassay described below. In the case of the Vaccinia virus, 1,000 OmriUnits represent a specific amount of antibodies in 1 ml. of plasma concentrate obtained from a population of 50 individuals vaccinated with the antigen that causes the production of specific antibodies in said individuals. It was shown that in the case of the Vaccinia virus, 20-40 OmriUnits will neutralize the Vaccinia virus in a neutralization test by approximately 50%. An example of such a neutralization test (PRNT50 = Reduction Plate Neutralization Test) is described below. Low volume unit dose form - a dosage form that can be administered to the patient in a single dispensation, usually by injection. In this way, the patient receives a complete individual treatment in a single and brief administration. A preferred volume is less than about 10 ml, preferably about 5 ml. or less, more preferable about 2 mi. Approximately 10% protein - between 8% and 12% protein concentration, preferably about 10%. A low aggregate content - less than 3% of protein aggregates in a solution, allowing the use of the solution for iv administration. Treatment of an infection - therapeutic treatment and / or prophylactic of the infection. In another aspect of the invention, a concentrated immunoglobulin composition is provided which includes specific antibodies immunoreactive with a pathogenic microorganism, characterized in that the specific antibody titer of the composition is at least 5 times higher, and preferably 10 times higher. , that the average titer of antibodies specific to a population of individuals previously vaccinated against one or more antigens associated with the pathogenic microorganism. The immunoglobulin products present such as Vaccinia immunoglobulin (VIG) are prepared using plasma from non-selected revaccinated donors. This results in a low specific immunoglobulin titer. Also, due to its low concentration, the prior art product should be administered by a slow intravenous infusion. In cases of intramuscular injections, the product (approximately 40 ml in volume) should be administered in several places and for several hours. The concentrated immunoglobulin composition produced, according to the invention, will sometimes be mentioned, with reference to a preferred embodiment in which the pathogenic microorganism is Vaccinia virus, such as HT-VIG (high titre vaccinia immunoglobulin), whereas the prior art immunoglobulin product will be mentioned sometimes, also with reference to this preferred representation as VIG (Vaccinia immunoglobulin). The method of the invention allows the selection of very high-grade plasma samples, resulting in a product of high potency and low volume. Blood or blood components of very high titre can be obtained repeatedly, being obtained from each of the high-grade individuals. In a preferred embodiment, the specific antibody titer of the concentrated immunoglobulin composition is at least 5 times higher, and preferably 10 times higher, than the average titer of the specific antibodies of a population of previously vaccinated individuals against one or more antigens associated with the pathogenic microorganism. The concentrated immunoglobulin composition preferably contains at least about 40,000 OmriUnits of the specific antibodies. In another preferred embodiment, the concentrated immunoglobulin composition contains at least about 10% protein. The high potency of the concentrated immunoglobulin composition allows it to be administered at very low volumes (iv or im) and therefore can be used in field conditions by any trained emergency personnel. As mentioned above, the prior art method for the treatment or prophylaxis of an infection by Pathogenic microorganisms are based on a VIG preparation produced by non-select plasma of revaccinated volunteers. The HT-VIG representation of the composition IVIg produced according to the invention has several advantages over the VIG currently in use. "It is expected that the HT-VIG is about 10 times more powerful than the regular VIG." The HT-VIG developed according to the invention can be administered by low volume IV or im route due to its high title and low added content. . Unlike the current VIG preparation that is administered by a physician trained by slow iv during hospitalization, the new product can be used in field conditions by any trained emergency personnel. • The new HT-VIG can be formulated in small, easy-to-use filled syringes or vials. "HT-VIG can also be used for the treatment of the adverse effects of Vaccinia vaccination and as a prophylactic or therapeutic treatment against smallpox in susceptible populations." After concentrating the plasma of selected individuals, the concentrate can be purified and / or concentrated by means of known techniques for the treatment of immunoglobulin solutions. Examples of such techniques are disclosed in U.S. Patent No. 6,468,733 and U.S. Patent No. 121,900, the contents of which are attached for reference. The method disclosed in the aforementioned US patent reduces the level of protein aggregates in the concentrated immunoglobulin solution and allows the final product to reach a level of 10% protein with a low level of aggregates for iv administration. For example, the purification method of immunoglobulins from a source solution such as Cohn Fraction II may consist of: (a) pretreatment of a cationic exchange resin with an acid solution whose pH is 4.0-4.5 (b) contacting the fountain solution with cationic exchange resin; and (c) extracting with solvents the immunoglobulins bound to the cation exchange resin. Prior to contact with the cation exchange resin, the source solution can be treated with an organic solvent and a detergent. The invention is illustrated with respect to the Vaccinia virus, however it can be practiced with respect to other pathogenic microorganisms, and especially other viruses. Examples of such microorganisms include West Nile Virus, anthrax, rabies, CMV virus, RSV virus, hepatitis B, influenza, and secreting microorganisms. botulinum toxin and other toxins. It is expected that an immunoglobulin composition according to the invention can be used not only to treat the adverse effects of vaccination, but also as a therapeutic or prophylactic treatment in case of an infectious epidemic. For example, in the case of smallpox, due to the risks of vaccination against smallpox, a significant proportion of the population should be excluded from any mass vaccination program, including children under 2 years of age, pregnant and lactating women, persons with weakened immune systems, such as patients with primary or secondary immunodeficiency, etc. In all the cases mentioned above, the need for passive immunization would be critical during a smallpox epidemic. The treatment with the immunoglobulin composition according to the invention can therefore be indicated for prophylactic immunization of this population. Alternatively, an immunoglobulin composition can be administered concomitantly with vaccination to minimize the risk of complications in these sub-populations.

BRIEF DESCRIPTION OF THE FIGURES To understand the invention and see how it can be done in practice, an exemplary representation thereof will be described below, by means of a non-limited example, with reference to the accompanying drawings in which: Figure 1 is a graph showing the distribution of the anti-Vaccinia titers of donor plasma revaccinated recently (30 days); and Figures 2 and 3 show fluctuations in the level of Vaccinia antibody titers in two high titers over a period of several months.

DETAILED DESCRIPTION OF THE INVENTION A. Preparation of immunoglobulin composition A representation of the preparation of an immunoglobulin composition according to the invention is detailed below: 1) Establishment of screening tools a. Development of an ELISA method: A linear, competitive ELISA assay was developed to determine the vaccinia antibody titer. This essay has 2 objectives: 1. Track donations of high-grade plasma; 2. Possible use of a post-vaccination evaluation tool. The ELISA assay is relatively fast and involves relatively few reagents. Like all banks blood for ELISA, the ELISA assay can be performed in a single dilution with only one duplicate.

Principle of the test The inactivated antigen of Vaccinia is bound to the surface of the microtiter wells. Samples in which they are suspected to contain specific immunoglobulin G (Vaccinia antibodies) such as serum, plasma, purified and concentrated immunoglobulins, calibration standards or controls are pipetted into wells and incubated at 372C. Vaccinia antibodies, if present, bind to the immobilized antigen. The unbound material is removed in the washing step. In the second stage a goat anti-human IgG alkaline phosphatase conjugate is adhered to the wells and the ELISA plate is incubated at 372C. After incubation, the unbound conjugate is removed by washing. The enzymatic activity of alkaline phosphatatase is then measured by a p-nitrophenyl phosphate substrate. The measurement is carried out in an ELISA reader at 405 nm. There is a direct relationship between the observed optical density and the antibody titre of Vaccinia.

Handling of samples The blood collection must be done according to the current practices. The serum or plasma should be separated as soon as possible to avoid hemolysis. Samples should be stored for 7 days at 28SC before use. For longer storage, freeze samples at -202C or colder temperature. Avoid repeated freezing and thawing. Samples with observable particles or after thawing must be centrifuged before the test.

Preparation of the washing solution One day before starting the ELISA procedure, prepare a working solution, adding all the contents of a bottle of 50 ml wash concentrate. (Rl) to 450 mi. of distilled and mixed water that rested overnight at room temperature until its complete dissolution. The prepared wash solution can be stored at 2-8 eC for up to 1 month. Prepare 500 mi. for a 12-strip plate.

Test procedure Sample and dilution of standards Using suitable test tubes and a microtitre plate, dilute the standard samples and controls as follows: a. Samples of the test: First, prepare a initial dilution of 1: 100 (sample of lOμl in a sample solvent R2 of 990μl). Then, continue the dilution of the samples at 1: 300 (150μl of a dilution of 1: 100 in a R2 test solvent of 300μl) and 1: 1200 (100μl of a dilution of 1: 300 in a test solvent R2 300μl). b. The standard calibrator (R3) should be diluted 1: 700, 1: 100, 1: 500, 1: 300 and 1: 600 in a Sample Solvent R2. Use an initial dilution of 1: 100 (calibrator of lOμl in diluent R2 of 990μl) and then dilute more to reach the above dilutions (50μl in 300μl, 50μl in 450μl, 50μl in 700μl, 50μl in 2950μl. Sample Solvent R2, respectively ). c. The negative standard control (R4) must be dissolved 1: 300: lOμl in 2.99 mi. of Sample Solvent R2. d. The positive standard control (R4) must be dissolved 1: 500: lOμl of R9 at 4.99 mi. of Sample Solvent R2.

ELISA test procedure 1. One pipette of 100μl of previously diluted samples, standard calibrator (calibrator), negative control (NegCont) and positive control (PostCont) in its respective microwells of the test strips according to the scheme below. Standard calibrators must be added in duplicates. A Sample Solvent (R2) is added in 2 wells used as blanks. 4 F G H Cover the strips with plastic cover and incubate at 37 aC for 1 hour. During the incubation, prepare the conjugate solution, diluting the concentrated conjugate in the Conjugate Solvent (R5). Dilute the conjugate according to the number of strips in use: Per one strip: 70μl in 930μl of solvent. For two strips: 140μl in 1860μl solvent, for three strips: 215μl in solvent 2800μl At the end of the incubation, remove the adhesive tape and using a plate washing device, empty all the wells by aspiration and wash them six times (200μl / well) with diluted washing solution. When completing the washings, make sure that all the wash solution was extracted. Distribute 200μl of the conjugate solution in each well. Cover the strips (see stage 2) and incubate at 37 eC for 1 hour. At the end of the incubation, aspirate the contents of the wells and wash as described in step 4. Quickly distribute 100μl of the substrate solution in each well. Cover the strips with aluminum foil and incubate for 45 minutes at room temperature. Stop the reaction by adding 50μl of Stop Solution (R8) to each well. Clean the bottom of the plate carefully. Read the absorbance (OD) of the wells at 405 nm. using an ELISA reader after 15 minutes of stopping the reaction. Note: Shake the strips well before reading.

Calculation and interpretation of results 1. Calculate the average DO value for all duplicates and subtract the OD reading from the average blank of all results. For the test to be valid, the following conditions must be met: (a) the control reading must be less than the reading of the 1: 700 dilution of the standard calibrator; (b) the average negative control reading should be less than the 1: 500 dilution reading of the positive control. 2. Plot the net OD values at 450 nm. obtained by the dilutions of the standard calibrator (on the Y axis) against the concentrations of these dilutions in OmriU / ml (on the X axis), where: the 1: 700 dilution corresponds to 2.1 OmriU / ml; the 1: 100 dilution corresponds to 1.5 OmriU / ml; the 1: 3000 dilution corresponds to 1.5 OmriU / ml; the dilution 1: 1500 corresponds to 1.0 OmriU / ml; the dilution corresponds to 0.5 OmriU / ml and the dilution 1: 6000 corresponds to 0.25 OmriU / ml. The linear range should be 0.3 - 1.5 OmriU / ml. 3. Determine the Vaccinia antibody titer of the test samples by interpolating the calibration curve and multiplying that value by the dilution factor of the sample. 4. Determine the control title of the positive (R9) according to the above. The whole procedure of the Test is valid only if the title of R9 is between 400 and 600 OmriU / ml.

Interpretation of results A subject immunized with Vaccinia can be classified into one of four categories: Very low responder: < 90 OmriU / ml Low responder: 90 - 440 OmriU / ml Medium responder: 450 - 1800 OmriU / ml High responder (excessively immune): > 1800 OmriU / ml Therefore and alternatively, the results can be interpreted, using the following table: Note: to. In most cases, only one of the two dilutions will result in a value within the linear range (0.3 - 1.5 OmriU / ml). However, in case both dilutions result in a value within the linear range, the sample should be considered as a low responder. b. The classification of vaccine response categories is tentative and based on the Israeli vaccination program. However, a population may respond differently in other vaccination programs and / or a different vaccine preparation. Therefore, the final scheme of interpretation will be determined according to the clinical study.

References: 1. Centers for Disease Control and Prevention, Vaccinia vaccine (smallpox): recommendations for the Advisory Committee on Immunization Practices (ACIP), 2001, MMWR Recomm. Rep. 2001 June 22, 50 (RR-10); 1-25. 2. Frey S.E., Ne man F.K, Yan L., Belshe R.B. (2003) Reaction to Vaccine against Smallpox in people immunized in the distant past. JAMA 289 (24): 3295-3299. 3. Henderson, D.A. (1999): Smallpox: clinical and epidemiological features. Infectious diseases Emerging - 5: 537-539. 4. Henderson, D.A. (1999): Smallpox as a Biological Weapon. JAMA 281 (22): 2121-2137. 5. WHO (2001) Smallpox. Weekly Epidemiological Report 76 (44): 337-344. 6. WHO - Recommendations for the Production and Control of the Smallpox Vaccine (2004). WHO - 53rd Expert Committee on Biological Standardization, February 17-21, 2003. Final Report.

The ELISA assay allows linear continuous quantification of the anti-vaccinia by generating a direct relationship between the optical density observed at 405 nm. and the concentration of antibodies Vaccinia in plasma and serum. 1 m of collected plasma derived from 50 vaccinated donors is used as a positive standard and a value of 1000 arbitrary units (= 1000 OmriUnits) is assigned, as described below in Example 1.

Validation of anti-Vaccinia antibody test equipment carried out as described below: 1. Internal test of accelerated stability, precision, sensitivity, specificity and recovery with negative, positive, standard and blood bank samples. This was carried out with at least three batches of product production (diagnostic equipment). 2. Adequate blood banks were hired to perform field validation tests. The teams of production lots were supplied to these establishments, which were trained in the place to operate the test equipment. The blood bank was required to test standard samples equipment and previously analyzed (positive and negative) to verify that the test equipment is properly operated. 3. After successfully completing paragraph (2) above, each blood bank was required to review 150 randomly chosen donor samples. These samples were checked in parallel to the reference test equipment. If the results of the test of the company's diagnostic test equipment and the reference tests agreed, the equipment was approved for the routine analysis of the blood units of the donors.

Ivalence with the serum neutralization method The gold standard for measuring biological activity (viral inactivation) of anti-viral antibodies is the viral neutralization assay. The neutralization of a virus is defined as the loss of infectivity by the reaction of the virus to a specific antibody. The loss of infectivity is achieved by the interference of a bound antibody with each of the steps leading to the release of the viral genome in the host cells. In this assay, the constant predetermined dose of the virus (400-600 PFU / ml) is allowed to react with other dilutions of the neutralized sample (plasma or intravenous immunoglobulin) and then inoculated into the cell culture. The Plate Reduction Neutralization Title 50% (PRNT50) is a specific and sensitive test that quantifies the dilution of the sample, resulting in a 50% reduction in the plates relative to the virus control well. This test is based on the fact that vaccinia virus produces cytopathic effects (ECP), which can be observed as plaques in cell culture. The ECP is neutralized by the presence of specific antibodies. In this test, a given viral load is mixed with a serial dilution of plasma samples or serum for a given time, after which the dilution series is cultured in the cell culture line of the host tissue of the virus. After 48-72 hours, the number of viral plaques is counted. The geometric average of the number of plates is calculated from the dilution series, as well as the viral count of the control plasma without the specific antibodies. Viruses are counted without the control result in a number called Neutralization Inhibition 50 - the dilution of plasma or serum resulting in a 50% reduction in viral titer. The test is very complicated, takes at least 4 days of work and is not adapted to high performance samples. It requires a fully equipped virological laboratory with the capabilities of continuous virus culture and cell culture in a highly classified control environment. Therefore, the equipment, the environment and technical capacity far exceed the conditions in conventional blood banks.

INSTRUMENTS AND MATERIALS 1. INSTRUMENTS 1.1 Pipettes of 1, 2, 5, 10 and 20 ml, sterile, for cell culture. 1. 2 6 wells of tissue culture plates. 1.3 Flasks of 75 cm2. 1.4 Incubators with water jacket at 37 SC with 5% C02 1.5 Filter system, cellulose acetate 0.22 μm. 1.6 Pump. 1.7 Laminar flow hood. 1.8 Filter 0.8 / 0.2 μm, 32 mm. in diameter, Supor (Gelman, product # 4658). 1.9 Microscope. 1.10 Water bath at 37 BC. 1.11 Plastic tubes. 1.12 Vortex 1.13 Chemical bell. 1.14 Incubator at 372C. 1.15 Folder filter. 1.16 Gauze bandage. 2. REAGENTS 2.1 Growth medium - MEM - from Earle, without L-Glutamine (Minimum Essential Medium Eagle, Biological Industries Beit Haemek, cat. # 01-025-1). .2 Fetal Calf Serum (FCS) (Biological Industries Beit Haemek, cat. # 04-121-1). .3 L-Glutamine in saline, 20 millimoles (Biological Industries Beit Haemek, cat. # 03-020-1). 2. 4 Penicillin (10000u / ml) - Streptomycin (100 mg / ml) Amphotericin B solution (25μ / ml) (Biological Industries Beit Haemek, cat # 03-033-1). 2.5 7.5% Sodium Bicarbonate Solution (Biological Industries Beit Haemek, cat # 03-040-1). 2.6 Solution of Amino Non-essential Acids (Biological Industries Beit Haemek, cat. # 01-340-1). 2.7 lOOmM Sodium Pyruvate Solution (Biological Industries Beit Haemek, cat # 03-042-1). 2.8 Trypsin EDTA solution (0.25% Trypsin) (Biological Industries Beit Haemek, cat # 03-050-1). 2.9 Dimethyl Sulfoxide (DMSO) (Sigma, cat # D2650). 2.10 Tragacanth gum (Sigma, cat # G-1128). 2.11 Ethanol (Riedel de Haen, cat # 32221). 2.12 Basic fuchsin, Pararosaniline (Sigma, cat # P1528). 2.13 Phenol (Sigma, cat. # P-4557). 2.14 Growth medium - MEM - (from Earle) 2X, without L-Glutamine (Minimum Essential Medium Eagle, Biological Industries Beit Haemek, cat. # 01-025-9). 3. CELLS AND VIRUSES BS-C-1 Cells: (ATCC - CCL-26) - Continuous liver epithelial cell line from Cercopi thecus aethiops (African green monkey), ATCC Lot # 1182351. Cells are maintained according to SOP # 08030105.

Vaccinia (strain IHD): (ATCC VR-156): Vaccinia belongs to the family of poxviruses, genus orthopoxvirus. Virion envelopes, slightly pleomorphic, ovoid or brick-shaped; 140-260 nm. in diameter, 220-450 nm. long. Composed of an outer layer consisting of lipid and tubular or globular protein structures, which encircle one or two lateral bodies and a nucleus that includes the genome. Virions contain a linear double-stranded DNA molecule.

PREPARATION OF SOLUTIONS Dilution medium - MEME 2% FCS The medium is prepared with MEM, adding 2mM L-Glutamine, lmM of sodium pivurate, 0. lmM of Amino Non-essential Acids, 1.5g / l of sodium bicarbonate, 1 % solution of Penicillin-Streptomycin-Amphotericin B and 2% FCS. The work is done under sterile conditions. 230ml of MEM, 2.5ml L-Glutamine, 2.5ml of sodium pivurate, 2.5ml of Amino Non-essential Acids, 5m / l of sodium bicarbonate, 2.5ml of solution of »Penicillin-Streptomycin-Amphotericin B and 5ml are added of FCS, respectively. Filter using a cellulose acetate filter system of 250 m. 0.2Dm. The medium is stable for one month at 2-82C if it is not contaminated. Before adding the medium to the cells, condition the medium to 372C by means of a water bath.

Coating medium - 2X MEME, 4% FCS The medium is prepared with 2X MEM, adding 4mM L-Glutamine, 1% Penicillin-Streptomycin-Amphotericin B solution and 4% FCS. The work is done under sterile conditions. 230ml of 2X MEM, 2.5ml of Penicillin-Streptomycin-Amphotericin B solution and lOml of FCS, respectively, are added. Filter using a cellulose acetate filter system of 250 m. 0.2Dm. The medium is stable for one month at 2-82C if it is not contaminated. Before adding the medium to the cells, add 2.4% sodium bicarbonate and condition the medium at 372C with a water bath.Covering medium - Tragacanth 4.3.1 Cleaning tragacanth Pour 20 g. of tragacanth for a laboratory glass with 60ml ETOH. Mix the tragacanth until a homogeneous suspension is obtained (15 minutes). Wait until decantation and discard the supernatant liquid. Repeat the mixture 3 times. Dry the tragacanth pill using the folder filter and then place it in the 37 SC incubator for 3 hours. 3 Pass it through a gauze bandage and store it at room temperature. 4.3.2 Working medium with tragacanth Dissolve 8 g. of tragacanth in 1000 ml of purified water for 2 hours at 702C. Achieve a volume of 1000 ml with purified water and autoclave at 1202C for 30 minutes. The tragacanth solution should be aliquoted to 50 ml vials. under sterile conditions. It can be stored up to 2 years at 2-8aC.

The medium is prepared with 2X MEM, adding 4mM L-Glutamine, 1% solution of Penicillin-Streptomycin-Amphotericin B and 4% FCS. The work is done under sterile conditions. 230ml of 2X MEM, 2.5ml of Penicillin-Streptomycin-Amphotericin B solution and lOml of FCS, respectively, are added. It is filtered through a 250 m cellulose acetate filter system. 0.2Dm. The medium is stable for one month at 2-82C if it is not contaminated. Before adding the medium to the cells, 2.4% sodium bicarbonate is added and the medium is conditioned at 37 aC by a water bath.

Solution for coloring - Fuchsin Prepare 5% Phenol by adding 50 ml Phenol to 950 ml of purified water. Prepare a solution of Fucsina 3% dissolving 3 g. of Fuchsin in 100 ml EtOH. Mix well the solutions of 100 ml of Fucsina and 1 L Phenol. Store at room temperature for up to 2 years. Protect from light with an aluminum foil.

. METHOD The duration of the neutralization test is 5 days and consists of the following stages: Day 1: Sow the BS-C-1 cells, which were cultured as described in POC (Operating Procedures) Common) # 08030105, on a 6-well plate (section . 1) . Day 2: Virus neutralization assay (section 5.2).

Day 5: Cell coloration and determination of PRNT50 (section 5.3). . 1 Sowing BS-C cells (Day 1) Sow BS-C-1 cells (passage 4-40) on a plate 6 wells at a concentration of 4x1O5 cell / well (according to POC # 08030105). The cells must reach confluence the next day for the virus neutralization test (day 2) . . 2 Virus neutralization test (Day 2) 5.2.1 Sample preparations . 2.1.1 Prepare a Vaccinia virus concentrate in a MEMS 2% FCS culture medium at a final concentration between 800-1200 pfu / ml. 5.2.1.2 Prepare serial dilutions of the sample in a growth medium, MEME 2% FCS, in a range of 1/40 - 1/40960. Plasma and serum samples should be inactivated at 56 aC for 30 minutes. 5.2.1.3 Mix each IVIG dilution with an equal volume of vaccinia concentrate, obtaining a final dilution of 1/80 - 1/81920 per IVIg and a concentration of 400-600 pfu / ml for Vaccinia virus concentrate (eg 500 μl of 1/40 IVIg diluted with 500 μl of vaccine virus 800-1200 pfu / ml vaccine virus resulting in a final IVIg dilution of vaccinia of 1/80 and 400-600 pfu / ml). . 2.2 Control of preparations Virus control: Vaccinia virus concentrate with culture medium 1: 1. Negative controls (no plate reduction): Medium control: Growth medium, MEME 2% FCS iVlg (for IVg assay): Mix OmriGam 5% batch # F18132 (produced by a plasma of origin North American) at a dilution of 1/40 with an equal volume of Vaccinia virus concentrate, obtaining a final dilution of 1/80 for OmriGam 5% and a final concentration of 400-600 pfu / ml for the Vaccinia virus concentrate. Plasma (for plasma assay): Mix a batch of negative plasma at a 1/40 dilution with an equal volume of Vaccinia virus concentrate, obtaining a final dilution of 1/80 for plasma and a final concentration "of 400-600 pfu / ml for the Vaccinia virus concentrate Serum (for serum assay): Mix a negative serum at a 1/40 dilution with an equal volume of Vaccinia virus concentrate, obtaining a final dilution of 1/80 for serum and a final concentration of 400-600 pfu / ml for the Vaccinia virus concentrate. Positive control (reduction of plates): Mix a positive sample at a dilution of 1/40 with an equal volume of Vaccinia virus concentrate, obtaining a final dilution of 1/80 for positive sample and a final concentration of 400-600 pfu / ml for the Vaccinia virus concentrate. Turn all IVIg mixes and controls for 90 minutes at 372C. 5.2.3 Inoculation of the virus (Day 2) . 2.3.1 After 90 minutes of incubation, observe the uni-molecular layers of BS-C-1 cells (prepared on day 1), which should be healthy and confluent. 5.2.3.2 Discard the middle of each well. 5.2.3.2 Carefully add 0.2 ml of the sample and control mixtures to the wells and gently shake the plate to obtain a uniform distribution of the inocula. 5.2.3.4 Let the unneutralized Vaccinia virus adsorb for 60 minutes at 372C in a C02 incubator. 5.2.3.5 Combine between the growth medium (MEM x2 4% FCS) and the tragacanth solution (1: 1) and add 2.4% sodium bicarbonate. 5.2.3.6 Add 3 ml of medium tragacanth to each well. . 2.3.7 Incubate the plate in an incubator at 372C for 72 hours.

Cellular staining and determination of PRNT50 (Day 5) 5.3.1 Examine the plate under the microscope for the observation of dissolved cells by means of lysines. . 3.2 The coloring process should no longer be sterile and should be carried out in a chemical campaign. 5.3.3 Empty the wells, hitting them slightly. . 3.4 Add 500μl EtOH to each well. Wait 2 minutes. Remove the solution from the wells, hitting them slightly. 5.3.5 Add 500μl Fucsin staining solution (Section 4) to each well. Wait 4 minutes. Remove the solution from the wells, hitting them slightly. 5.3.6 Count the number of plates. 6. ACCEPTANCE CRITERIA FOR THE TEST - Virus control should result in 80-120 PFU / well. The negative control should result in a uniform and intact uni-molecular layer. Plasma, serum and negative IVIg controls should provide > _ 80% of virus control plates. - The positive control must supply < 50% plates compared to plasma, serum or negative IVIg control. 7. EVALUATION OF THE RESULTS The PRNT50 (expressed in μg / ml) for each sample is of IgG concentration in which there is a 50% neutralization of the Vaccinia virus, as determined by the non-linear relationship (dose-response curve) between the record of igG concentration (initial IgC concentration divided by dilution) and percentage of plaque neutralization relative to the control sample (taken as 100%). 8. REFERENCE Virology Methods Manual, Brian W.J. Mahy and Hillar 0 Kangro. 4) Pilot study A pilot study was carried out with the Israeli Defense Forces (IDF), which served as a model for the vaccination and selection of plasma donors. After a complete analysis of the data, the statistical power was generated from which the exception and exclusion criteria for the plasma donation were finally formulated, in addition to a general blood bank. Vaccination of IDF subjects was carried out in accordance with the guidelines of the Center for Disease Control and Prevention (CDC) (http: //www.bt.cdc.gov/ agent / smallpox / response-plan / index. asp # guideb). Additional criteria that may arise from the complete analysis of the pilot study were implemented in the vaccination protocol. Plasma screening was preformed using the same standards as in the pilot study, with some changes derived from the complete statistical analysis of the data. High-titre hyper immune donation (HT) was finalized after fully evaluating the variability of the titer and estimating the percentage of the population that will contribute to the hyperimmune HT plasma. An example of Donor variability is shown in Figure 1. This hyperimmune HT population was maintained between 10-15% of all subjects who presented either seroconversion or clinical signs that the immunization was successful. The plasma collection was carried out according to the FDA guidelines and the standard operation of the local blood banks. Selected subjects were repeatedly used as, for example, using plasmapheresis or plasma donation technique (see, for example, US Patent No. 6,692,739, the contents of which are incorporated herein by reference). Subjects without clinical signs were excluded from plasma donation. These subjects were revaccinated. The clinical signs are the following. A normal primary vaccination appears as a papule after 3-4 days and quickly progresses to a blister, with erythema surrounding the fifth-sixth day. The center of the blister sinks and progresses into a well formed pustule on the eighth-ninth day. On the twelfth day or soon thereafter, the pustule withers, forming a brown scab that progresses from the center of the pustule to its circumference. After 2.5 to 3 weeks, the scab falls off and a well-formed scar remains.

Table 2: Normal reaction times to Vaccinia vaccine Rarely, in some individuals not previously vaccinated, the vaccination techniques considered appropriate may not cause any reaction. It must be assumed that the subject is not immune and repeat attempts to achieve a primary intake. At least three attempts have to be made, changing the cutaneous sites after a second failed attempt. Systematic symptoms: The expected systemic symptoms normally occur after a week of vaccination are the following: "Pain in the vaccine site. • Severe erythema surrounding the vaccination site." Upset.

• Lymphadenopathy (local) • Myalgia, headache, chills, nausea, fatigue.

• Fever. The occurrence of these normal reactions varies considerably from one study to the other. The following table lists the symptoms listed in the studies and provides an indication of their range: Table 3: Occurrence of reactions to Vaccinia vaccine The donation of plasma should be carried out when the scab of the vaccine is separated from the skin, or 27 days after the vaccination, according to the last one. The plasma samples were rated according to the FDA guidelines for plasma safety. All qualified donations are tracked for high Vaccinia title. Only the donation of plasma with minimum Vaccinia titles predetermined will be concentrated for the fractionation of HT hyperimmune VIG. The plasma donation receipt and the fractionation discharge was according to the acceptance criteria prepared in advance. These criteria include only the use of plasma units with antibody titers of Vaccinia about 1800 OmriUnits / ml. The production of hyperimmune HT VIG lots. to. Re-suspension of 10-12 kg. of paste II (equivalent to 2.5-3.0 kg of immunoglobulin). b. Production of 10% mass, treated solvent detergent and 20 nanometer immunoglobulin filters. c. Introduction of the product in syringes previously filled with 2 ml. Hyperimmune HTV VIG can be characterized according to the USP monograph for intramuscular immunoglobulin. The stability studies of the hyperimmune HT VIG final packaging should also be carried out. The following is a characterization of the product of an example immunoglobulin composition (HT-VIG) according to the invention.

Composition HT-VIG is a sterile solution containing 10% protein (100 mg in 2 ml of solution of which at least 95% is Human Normal Immunoglobulin G), 10% maltose and water for injections.

Description of the production process The HT-VIG is obtained by fractionation of Cohn cold plasma ethanol.

Safety The HT-VIG is subject to Viras extraction / inactivation technology, composed of 3 stages of inactivation: Cohn fractionation of cold ethanol was validated as the inactivation stage of the primary virus. The HT-VIG goes through a second stage of inactivation of the virus by the Solvent Detergent method using TnBP / Triton X-100 and a third stage of inactivation by nano-filtration at pH 4 and converts the HT-VIG into a highly immunoglobulin. safe.

Pharmaceutical form HT-VIG is a clear or slightly opalescent liquid, almost odorless, colorless to pale yellow for intravenous or intramuscular administration.

Type of vial The sterile filling of the product is carried out in glass vials, siliconized 10 ml. with 4-5 my filling volume or in syringes previously filled with 4-5 ml.

Storage conditions Syringes / vials should be stored at a temperature between 2-82C, protected from light. 2. Specific information B. Data Collection, Analysis and Interpretation Data collection is a simple process that can be performed according to the Common Operating Procedures (POC) of the local database that are already implemented in each center. The questionnaires of the subjects for the vaccination and evaluation of the tracking program were formulated. The instructions for collection, storage and shipment of blood were written and supervised according to POC. All the annexes to the vaccination and plasma collection tracking protocols were formatted in both electronic and printed copies to allow analysis and supervision by the quality assurance team. The data was entered into a database system on a personal computer (such as Paradox) to enable online consultations and production of updated reports. All statistical analysis was carried out using a statistical software package for personal computer (like being SAS). Standard parametric tests were performed to compare symptomatic and antibody responses. The primary analysis included safety, serological and clinical data. The parameters of response to the vaccine included data of clinical cutaneous reactions, normal reactions, adverse effects, severe adverse effects and anti-Vaccinia titers. The subjects who possessed Titles higher than the edge selected as eligible for the collection and assigned for hyper immune, were included in the preliminary analysis but were also analyzed separately in a sub-analysis to see if they correlate to any of the parameters collected in the questionnaire of vaccination.

C. Security Viral extraction / inactivation technologies are known. For example, such a technology is disclosed in U.S. Patent No. 6,468,733, the contents of which are incorporated herein by reference. The HT-VIG product may include stages of viral extraction according to these technologies, such as nano-filtration and treatment by solvent detergent, which exhibits high margins of viral inactivation of all known viruses, including Parvo-virus. The detergent-solvent was extracted by means of a specific solvent detergent extraction resin that has the ability to bind hydrophobic substances and molecular sieve of large molecules. The flow of the column produces an immunoglobulin solution with very low content of dimer and polymers, allowing a high yield through nano-filtration. This results in a preparation of liquid immunoglobulin lacking active viruses, presenting a very high production. Also, the process mentioned previously it can be performed at a low pH, thus also allowing the inactivation of sensitive viruses at low pH. The storage of the liquid product can be at room temperature for at least 2 years in the syringes previously filled and ready for use. For longer periods of storage, the product remains stable for up to 3 years at 2-8aC.

D. Results Three studies related to plasma donation samples from Israel and the USA were carried out: The plasma for fractionation and production of hyper immune Immune Vaccinia is generally collected from voluntary revaccinated donors. To begin the investigation, the variability of Vaccinia immunoglobulin in plasma donations and the percentage of plasma units suitable for the concentration were studied. The collection of appropriate plasma donations from revaccinated subjects is based on the clinical manifestation of the vaccine. On the other hand, the hyper immune plasma collection is based primarily on plasma titers. The correlation between these two methods was evaluated. The production of hyper immunoglobulins carries inherent risks of achieving low yields that can reduce drastically the economic viability of a certain production technology. The ELISA assay, being highly sensitive, can detect residual titers of anti-Vaccinia IgG in regular plasma concentrates. It can also detect these G immunoglobulins in batches of intravenous immunoglobulins. The theoretical yield of the VIG production process was calculated. The results indicate that the method of the invention is suitable for the production of high titre VIG.

Example 1: Tracing of anti-vaccinia titers in plasma units donated by revaccinated subjects. Background: Plasma donations for the production of intravenous immunoglobulin rich in Vaccinia were collected from revaccinated donors during the Israeli emergency program in which military personnel and selected physician was vaccinated against smallpox. Because the selection of donors was solely based on signs of clinical manifestation and not on serum immunoglobulin-conversion titers, an estimation was necessary to evaluate the titers in the plasma production concentrates.

Method: 37 plasma donations were randomly selected from 24,000 subjects who were inoculated via intradermal 30 days before with 20μl of Vaccinia virus Elstree (average title, 107 pfu per millimeter). The plasma units were checked for anti-Vaccinia immunoglobulin titers, by the ELISA assay. 3 ml of each plasma donation were mixed with the other samples and the concentrate served as the positive standard. The positive standard of concentrated plasma was then assigned 1000 arbitrary units (1000 OmriUnits / ml). 20-40 OmriUnits will neutralize the Vaccinia virus by 50% in the neutralization test described above (PRNT50 trial). The positive plasma standard of Vaccinia concentrate can be obtained on request at Omrix Biopharmaceuticals Ltd., Tel Aviv, Israel.

Results: The results are summarized in Figure 1. It was found that the variability of donated plasma units varies between 26 units and 6530 OmriUnits. The calculated average was 971/8 OmriUnits, the standard deviation was 1349 and the average was only 542 OmriUnits. The results of the calculated average coincide with the average of the plasma units experimentally mixed, indicating that the mixture of different plasma units does not differ from the calculated one. This indicates that the ELISA assay does not suffer from inhibitory reactions that sometimes occur when mixing plasma donations.

Conclusion: Only 10% of the population has titles that are significantly higher than the average of the plasma donation collected from the revaccinated subjects. It is apparent that if only donations possessing titres greater than 1800 OmriUnits / ml are collected (very high plasma donations), the hyperimmune concentrated plasma titer will be at least 5 times greater than the regular immunoglobulin rich in Vaccinia collected . Therefore it is obvious that the screening of plasma donation from the entire revaccinated population is required to identify individuals with very high titres, who possess a very high specific antibody titer. It was discovered that individuals with very high titre stably maintain a very high specific antibody titer. Figures 2 and 3 show the titles of two individuals with high titers of approximately 3000 OmriUnits / ml. This title was maintained even 3 months after the revaccination and after almost 20 plasma donations.

Example 2: Correlation between clinical responses and seroconversion of smallpox vaccination in revaccinated adults. Background; The production of Vaccinia immunoglobulin (VIG) is based on the plasma collection of recently vaccinated adult subjects. Recent reports they indicate that the clinical response of naive subjects exceeds 95% compared to reports of the early 60s that showed that the clinical response of revaccinated children is much lower. The clinical experiment was aimed at evaluating the response percentage and estimating the correlation between the clinical response of the revaccinated subjects and the increase in anti-Vaccinia titers.

Method: In this prospective, randomized, single-blind study, 157 adults who were previously immunized were inoculated intradermally with 20μl of the Elstree Vaccinia virus (mean titer, 107 pfu per millimeter), using a hypodermic syringe and the site was covered with semi-permeable bandages. The subjects were controlled for blistering on the third-fourth day and the seventh-ninth day (clinical response to the success of the vaccination) and for adverse events for 30 days after the immunization. The serum samples were taken on day 0 (before vaccination), day 14 and day 30 (plasma donation). The serum titers were examined using quantitative, linear ELISA.

Results: Success rates were slightly different between clinical responders and seroconversion. The clinical responders were 62.87% - very similar to the subjects who demonstrated seroconversion (62.42%). Serum titers in all subjects were reduced by an average of 30% between days 14 and 30 after revaccination. In addition to the formation of pustules, common adverse events consisted of the presence of an acute viral disease, including the formation of satellite lesions, regional lymphadenopathy, fever, headache, nausea, muscle aches, fatigue, chills, which appeared only in a small percentage of subjects. The correlation between the sero-converted subjects (results of day 14 versus day 0) and the clinical formation of pustule appearance in the seventh-ninth days was 87.74% - very close to the percentage found among the sero-converted subjects who had pustules on days 3-4 and days 7-9 (88.44). The serum titers on day 0 were highly variable and became even more variable as the titres increased after 14 days. This variability also appeared 30 days after vaccination. Only 10-15 percent of the subjects possessed high titers that would be suitable for the production of hyperimmune immunoglobulin Vaccinia.

Conclusion: When 20ul of the Stree vaccine strain at a titre of, 107 pfu were administered to revaccinated adult subjects, 62.87 responded clinically, similar to the 62.42 seroconversion indices. There is a very high correlation between the two methods (approximately 88%). The Anti-Vaccinia titles reach their maximum point 14 days after vaccination and declines by approximately 30% at 30 days. The titles of the revaccinated subjects were highly variable at 30 days after vaccination and only 10-15% of the revaccinated subjects had titles over 1000 Arbitrary units that comply with the production of hyper immune vaccine globulins. These data suggest that a hyperimmune subject tracking test is mandatory.

Summary Preliminary studies indicate that the immunoglobulin G titers of the revaccinated volunteers are highly variable, thus the VIG products obtained from such concentrated plasma result in relatively low titres. Studies indicate that it is feasible to use the rapid ELISA screening method in which high titre anti-vaccinia samples can be selected. Plasma units of a high concentrated titre corresponding to 10-15% of the donor population for the production of intravenous immunoglobulin of 10% and condensation of the concentrate will result in the production of HT-VTG. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (19)

1. CLAIMS Having described the invention as above, the cnt of the following claims is claimed as property: 1. A method for the preparation of a concentrated immunoglobulin composition for treating subjects vaccinated against or infected with pathogenic microorganisms, characterized by comprising: (a) selecting a population of individuals previously vaccinated against one or more antigens related to the pathogenic microorganism; (b) determining the level of specific antibodies immunoreactive with said pathogenic microorganism in blood or blood component of said individuals, to identify individuals with very high titers, which possess a very high titer of said specific antibodies; (c) combining blood or blood components cining immunoglobulins of said individuals with very high titers; and (d) purifying and / or concentrating the product of step (c), to obtain a concentrated immunoglobulin composition.
2. 2. The method according to claim 1, characterized in that it also consists in the step of aliquoting the concentrated immunoglobulin composition in a low volume unit dosage form.
3. The method according to claim 2, characterized in that the low volume unit dose form includes a previously filled vial or syringe.
4. 4. The method according to any of claims 1 to 3, characterized in that the pathogenic microorganism is a virus.
5. 5. The method according to claim 4, characterized in that the virus is a vaccinia virus.
6. The method according to any one of claims 1 to 5, characterized in that the titer of the specific antibodies of the concentrated immunoglobulin composition is at least 5 times higher than the average titer of the specific antibodies of said vaccinated population.
7. The method according to claim 6, characterized in that the titer of the specific antibodies of the concentrated immunoglobulin composition is at least 10 times higher than the average titer of the specific antibodies of said vaccinated population.
8. 8. The method according to any of claims 1 to 7, characterized in that the composition of concentrated immunoglobulin cins at least about 10% protein.
9. The method according to any of claims 1 to 8, characterized in that blood or blood components are obtained from each of said high-grade individuals.
10. The method according to claims 1 to 9, characterized in that the blood component consists of plasma.
11. 11. An immunoglobulin composition, characterized in that it is prepared according to the method of any of claims 1 to 10.
12. 12. The immunoglobulin composition according to claim 11 with a low aggregate cnt.
13. The immunoglobulin composition according to claims 11 to 12, characterized in that they comprise specific anti-vaccinia antibodies at a titer greater than or equal to 40,000 OmriUnits / ml when measured using the ELISA assay in which a positive standard Concentrated plasma prepared from a population of previously vaccinated individuals against one or more antigens associated with the pathogenic microorganism is assigned the value of 1000 OmriUnits / mi.
14. 14. The immunoglobulin composition characterized in that it is in accordance with any of claims 11 to 13 comprising about 10% protein.
15. 15. A previously filled vial or syringe, characterized in that it cins a low volume unit dose of the composition according to any of claims 11 to 14.
16. 16. Use of a composition according to any of claims 11 to 14, to develop a medication to treat or ameliorate symptoms associated with adverse reaction to viral vaccination.
17. 17. Use of a composition according to any of claims 11 to 14, containing specific anti-vaccinia antibodies, to make a medicine to treat or prevent smallpox infection.
18. 18. A concentrated immunoglobulin composition comprising specific immunoreactive antibodies with a pathogenic microorganism, characterized by a specific antibody titer of the composition, being at least 5 times higher than the average titer of the specific antibodies of a population of individuals previously vaccinated against one or more antigens associated with the pathogenic microorganism.
19. 19. The concentrated immunoglobulin composition according to claim 18, characterized in that the average titer of the specific antibodies is at least 10 times higher than the average titer of the speci fi c antibodies.