Abstract:
Precipitates soluble in aqueous solution and suitable for stimulation and/or transfer of cell-mediated immunity in mammals and other animals is disclosed together with a solvent extraction process for obtaining this desirable product from crude bovine transfer factor. Lymphocytes from lymph nodes and blood of donor cattle are employed in formulating a Bovine Transfer factor by incubation release procedure. The fractionation of Bovine Transfer factor yields three precipitate fractions by the use of organic solvents. A relatively high incidence of transfer with the precipitates yields excellent immunity to certain diseases in animals.

Description:
BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     This invention relates to aqueous soluble precipitates that can stimulate or transfer cell-mediated immunity and to the production thereof from unfractionated bovine transfer factor. 
     (2) Description of the Prior Art 
     The prior art teaches that it is not possible to transfer cell-mediated immunity (delayed skin hypersensitivity reaction) with serum. However, viable lymphocytes taken from a reactive person and transferred to a non reactive person will temporarily make the recipient reactive, that is, tuberculin-positive. In man--but not in animals--it is also possible to transfer reactivity of &#34;delayed&#34; reactivity by means of nonviable extracts from lymphocytes. This means, this material, has been referred to as &#34;transfer factor&#34;. See Lawrence, H. S. J. Clin. Invest, 219, 34 (1955) and Lawrence H. S. in &#34;Transfer Factor: Basic Properties and Clinical Applications&#34;, p. 741 (Ascher, Gottlieb, and Kirkpatrick editors), Academic Press, New York, 1976. 
     In the prior art Phillip H. Klesius, et al., in Transplanation Proc. VII(3), 449-452, 1975, disclosed the first isolation of transfer factor from cattle lymphocytes. The bovine transfer factor in crude form, stimulated or transferred cell-mediated immunity across species barriers. 
     The literature discloses that unfractionated bovine transfer factor stimulates or transfers cell-mediated immunity against certain diseases in animals. The efficiency of the unfractionated bovine transfer factor was limited. [See &#34;Bovine Transfer Factor, Isolation and Characteristics&#34; by Phillip H. Kleusius, et al., in &#34;Transfer factor: Basic Properties and Clinical Applications,&#34; p. 311 (Ascher, Gottlieb, and Kirkpatrick Editors) Adacemic Press, New York, 1976.] 
     The teachings of the prior art characterized bovine transfer factor as having properties and characteristics similar to that of human transfer factor. The disadvantage of human material obtained from a single donor in comparison to larger yields obtained from single donor cow. See Klesius, P. H., et al., Clinical Immunology and Immunopathology, 7, p. 240-252, 1977. 
     There is no known commercial method for the solvent extraction of bovine transfer factor to produce active precipitates with all the characteristics of the present invention. The prior art teaches that liquid chromatography and electrophoresis have yielded small amounts of transfer factor which makes these methods impractical to use as a means of commercial extraction. 
     The Review of Medical Microbiology, 11th edition, compiled by Drs. Ernest Jawetz, Joseph L. Melnick, and Edward A. Adelberg, provides a brief review of the various facets of pertinent data which would enlighten the reader of the present specification. 
     SUMMARY OF THE INVENTION 
     Lymphocytes obtained from specific area of the lymphatic system of donor cattle are ground, then separated to discard the unwanted solids and utilize the liquid portion in the preparation of a culture. The lymphocytes contained in this liquid portion are incubated in Hank&#39;s balanced salt solution. Cell viability is verified by trypan blue exclusion, and the viability would decrease by 60% to 70% during the incubation period. The incubated material is separated by centrifugation. The supernatant culture fluid is dialyzed against deionized water; the dialysate is lyophilized; and the resulting dry material is designated &#34;unfractionated Transfer Factor, (TF)&#34;. 
     The improvement of the process begins when the TF is dissolved in water, the pH adjusted to about 6.0 to 6.5, and fractionated with alcohol. All steps of fractionation are carried out throughout at about 4° C. Alcohol is added dropwise to form an alcohol-water interface, which is fibrous. The mixture is stirred and the precipitate removed. The supernatant fluid is separated and the pelleted precipitate drained and dissolved in water, and lypholized to obtain Fraction I. 
     Alcohol is added to the supernatant, dropwise, as before, to obtain a second interface, which is then stirred and separated as before, thus yielding a precipitated Fraction II. The combined two precipitates, I and II fractions, are useful in intraperitoneal and intramuscular subcutaneous injections and oral ingestion to stimulate or transfer cell-mediated immunity in mammals. 
     The fractions I or II alone were active in stimulating cellmediated immunity. Although, it appears that the fraction II precipitate contains most of the material responsible for transfer factor activity, fraction I is also active in stimulating cellmediated immunity. 
     The primary object of the present invention is to provide a safe, nontoxic, microbial-free fraction of transfer factor that has a high rate conversion or efficiency (80% or better) for causing cell-mediated reactivity in mammals. The alcohol fractionation procedure provides a product that has not been denatured and is soluble in aqueous or physiological solution for treatment of mammals by multi-routes. 
     Another object is to provide a large amount of fractionated transfer factor that can be rapidly prepared from cattle that have been specifically sensitized for cell-mediated immunity against certain diseases. Still another object is to provide a solvent extracted transfer factor to be produced continuously on a commercial scale from the donor cattle and use in mammals to stimulate their cell-mediated immunity against the disease to which the cattle were sensitized. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In the practice of the present invention, prior art fractionation techniques, such as liquid chromatography and electrophoresis yielded lower amounts of transfer factor that may be contaminated with organic solvents toxic to mammals. 
     The bovine transfer factor is prepared from cattle sensitized for specific cell-mediated reactivity against the immunizing antigen(s). Blood or node lymphocytes are separated from unwanted tissue or debris. 
     The lymphocytes are cultured in Hank&#39;s balanced salt solution for a period of at least 4 hours at 37° C. By means of this incubation-release method the transfer factor material is separated from the lymphocytes (90% or better). 
     The unwanted large molecular (&lt;15,000) weight material of the resulting cell-free culture supernatant is removed by dialysis against deionized water or by ultrafiltration. Following this treatment the unfractionated transfer factor is extracted by the solvent step, using food-grade solvent such as ethenol is suitable. A typical solvent to transfer factor ratio for the first precipitate, Fraction I, is about 2 to 1 volumes. Extraction temperatures usually are about 4° C. For the second precipitate, Fraction II, the ratio is usually 4 to 1 volumes. 
     The final product is white or colorless and nontoxic for mammals. The product is soluble in aqueous or physiological solutions and is very active in stimulating or transferring cell-mediated immunity specifically or nonspecifically to mammal recipients. 
     The present invention generally is applicable to all mammalian species of animal including man. 
    
    
     The following example is provided to illustrate the process and product of this invention, and should not be construed as limiting the invention in any manner whatever. 
     EXAMPLE 
     Animals: 
     Calves 4 to 6 months of age were housed in individual pens or on concrete enclosures and maintained under management practices devised to prevent parasitic infection, especially coccidiosis. 
     Bovine donors for dialyzable transfer factor (TF d ) 
     Selection of donor cattle was based on positive skin reactions and in vitro lymphocyte stimulation responses to the test antigens. TF d  donors for E. bovis antigen and Purified Protein Derivative (PPD) were first infected with 100,000 E. bovis oocysts and then sensitized by subcutaneous injection of Mycobacterium bovis sensitinogen (USDA). Donors for PPD were given only M. bovis sensitinogen. Keyhole limpet hemocyanin (KLH) TF d  donors were given 8 mg of KLH in Freund&#39;s complete adjuvant. About 30 days after sensitization the donor animals were tested for Cell-mediated immunity (CMI) reactivity against the test antigens. Skin reactions greater than 9.0 mm in diameter at 48 hr and lymphocyte stimulation indexes (SI) greater than 1.50 (&gt;50% above controls) were considered positive for donor selection. 
     TF d  Preparation 
     Donor cattle were killed, then the mesenteric, thoracic, submandibular, cervical, auxillary, prefemoral and popliteal lymph nodes were removed from donor cattle, Crude TF d  was isolated from the tissue according to the procedure previously described. Briefly, TF d  was prepared by incubation of the lymphocytes in Hank&#39;s balanced salt solution for a period of 4 hr. at 37° C. Cell viability by trypan blue exclusion decreased by 60 to 70% during this incubation period. Following centrifugation the supernatant culture fluid, free of cells and cellular debris, was dialyzed against deionized water for 16 to 18 hr at 4° C. The dialysate was lyophilized, and the resulting dry material was designated &#34;unfractionated TF d  &#34;. Approximately 10 6  lymphocytes yielded 2.0 mg (Dry wt. of TF d ). The dose of TF d  for a 74-kg recipient animal was 400 mg (the amount extracted from 2.0×10 8  lymphocytes). The TF d  was dissolved in 5 ml of deionized water (ph 6.0 to 6.5) and given by intraperitoneal injection. 
     Alcohol-fractionated TF d   
     TF d  was fractionated with 95% ethanol by first dissolving the TF d  at a concentration of 50 mg/ml in deionized water and then chilling the TF d  solution at 4° C. All steps in the fractionation procedure were done at 4° C. from this point. Two volumes of alcohol were added dropwise to the TF d  solution so that an alcohol-water interface was developed. After gentle mixing by stirring, the first precipitate developed immediately. It was fine and fibrous, or strand-like, in appearance. At the end of 1 hour, the precipitate was removed by centrifugation (700 g for 15 min). The supernatant was carefully separated from the precipitate by decantation. The pelleted precipitate was thoroughly drained to remove residual alcohol and then dissolved in deionized water and lyophilized. 
     Two volumes of ethanol were added to the supernatant, and the solution was mixed by gentle stirring. Precipitate was visible within an hour after the addition of the alcohol but appeared to require a longer precipitation time. After overnight (14-16 hr) extraction, the precipitate was collected by centrifugation and separated from the supernatant. The drained precipitate (designated &#34;second precipitate&#34;) was dissolved in deionized water and lyophilized. In contrast to the first precipitate, the second was not fine and fibrous but more flaky and amorphous in appearance. Also, after centrifugation the second precipitate had a greater tendency than the first to stick to the glass wall of the centrifuge tube. 
     The supernatant from the second precipitate was lyophilized after removing the second precipitate. In Transfer experiments, the three fractions of the TF d  were tested at concentrations equivalent to 400 mg of unfractionated TF d . The three fractions were tested for CMI transfer as combined precipitates, second precipitate only, and supernatant. 
     Antigen and skin tests 
     E. bovis antigens were prepared as previously described from oocysts. PPD was prepared from Mycobacterium bovis at the Veterinary Service Laboratories, Animal and Plant Health Inspection Service, USDA, Ames, Iowa. KLH antigen was obtained from Dr. Dennis Burger, Veteran&#39;s Administration Hospital, Portland, Oregan. Cattle were skin tested by intradermal injection of 0.1 ml. in the cervical region and skin reactions were read at 24 and 48 hr. The skin test doses used were 800 μg for E. bovis antigen, 16 μg for PPD, and 350 μg for PPD, and 350 μg for KLH. The area of the test site was rotated when animals were tested more than once. 
     Lymphocyte stimulation assay 
     The semimicro protein synthesis assay for early detection of blastogenesis was used as previously described. Lymphocytes were obtained from peripheral blood using Ficoll-sodium metrizoate. Lymphocyte cultures were plated at a concentration of 2.0×10 5  cells/well and incubated at 37° C. for 36 hr. in 5% CO 2 . After the incubation, 1 μCi of  3  H-leucine was added to each culture. After 4 hr the cultures were harvested with a semiautomatic multiple sample processor. The incorporation of  3  H-leucine was measured in a scintillation spectrophotometer and was expressed as counts per minute (CPM) for quadruplicate cultures. The stimulation index (SI) reported was obtained by dividing the average CPM with antigen by the average CPM of control cultures without antigen. For each antigen test, at least 4 concentrations ranging from 6.0 to 0.1 mg/culture were used in the blastogenesis assays to measure the optimal SI. Student&#39;s t test was used for determining the statistical differences between test and control cultures. 
     CMI transfer and assay schedule for TF d   
     Within 5 days after CMI assay (pretest), TF d  recipients were treated. The pretest CMI assay was measured to determine the existence of immunity to test antigens before TF d  treatment. Stimulation of CMI against test antigens was determined 7 days after TFd treatment by CMI assay. The effect of TF d  treatment was determined from the results of this assay (test). 
     The material produced by the process of this invention was tested in the manner indicated on Tables I, II, and III, which tables having originated in the &#34;Bovine Transfer Factor: In Vivo Transfer of Cell-Mediated Immunity to Cattle with Alcohol Precipitates&#34; by Phillip H. Klesius and H. Hugh Fudenberg, being publication No. 101 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Table I presents the data applicable to &#34;unfractionated&#34; TF, and Table II presents the corresponding data applicable to the alcohol fractionated work of the present invention. The data herein was obtained from cattle which were previously skin tested. Table III, unlike the previous two deals with the transfer of Cell-Mediated Immunity with alcohol fractionated Transfer Factor. 
     
                                           TABLE 1.__________________________________________________________________________TRANSFER OF CMI WITH UNFRACTIONATEDTF.sub.d IN CATTLE PREVIOUSLY SKIN TESTED       Before TF.sub.d.sup.a                        After TF.sub.d.sup.b                Skin reaction:  Skin reaction:       Blastogenesis:                diameter of                        Blastogenesis:                                diameter of       stimulation                induration                        stimulation                                induration       index    (mm)    index   (mm)Treatment Animal No.        E. bovis            PPD.sup.d                 E. bovis                     PPD                        E. bovis                             PPD                                E. bovis                                     PPD__________________________________________________________________________ 1     1.09 1.14                0.0  0.0                        1.87.sup.e                             3.65.sup.e                                9.0  11.2Transfer 2     0.91 1.01                0.0  0.0                        5.88.sup.e                             4.99.sup.e                                9.1  6.6Factor 3     1.01 0.95                0.0  0.0                        3.44.sup.e                             1.69.sup.e                                12.8 12.4 4     0.98 0.96                0.0  0.0                        1.40.sup.e                             1.26.sup.g                                13.0 0.0 5     1.00 0.96                0.0  0.0                        1.06.sup.f                             1.60.sup.e                                12.3 13.0 6     0.97 0.89                0.0  0.0                        1.03 1.05                                0.0  0.0 7     1.00 1.05                0.0  0.0                        1.08 1.04                                0.0  0.0None  8     1.04 1.09                0.0  0.0                        0.99 0.94                                0.0  0.0 9     1.01 1.04                0.0  0.0                        1.00 0.84                                0.0  0.0__________________________________________________________________________ .sup.a Test reactions 5 days before TF treatment .sup.b Test reactions 7 days after intraperitoneal injection of 400 mg of TF .sup.c E. bovis  antigen: Optimal antigen concentration for blastogenesis and 800 μg antigen for skin test. .sup.d Purified protein derivative: Optimal antigen concentration for glastogenesis and 800 μg antigen for skin test. .sup.e p &lt;0.01 .sup.f Not significant 
    
     
                                           TABLE II__________________________________________________________________________TRANSFER OF CMI WITH ALCOHOLFRACTIONATED TF.sub.d IN CATTLE PREVIOUSLY SKIN TESTED        Before TF.sub.d.sup.a                         After TF.sub.d.sup.b                 Skin reaction:  Skin reaction:        Blastogenesis:                 diameter of                         Blastogenesis:                                 diameter of        stimulation                 induration                         stimulation                                 induration        index    (mm)    index   (mm)Treatment  Animal No.         E. bovis             PPD.sup.d                  E. bovis                      PPD                         E. bovis                              PPD                                 E. bovis                                      PPD__________________________________________________________________________  11    1.00 1.08                 0.0  0.0                         1.44.sup.e                              1.34.sup.f                                 10.0 8.0Alcohol  12    1.04 1.00                 0.0  0.0                         1.40.sup.e                              6.57.sup.e                                 9.2  7.0PrecipitatesCombined  13    1.00 1.00                 0.0  0.0                         1.72.sup.e                              2.93.sup.e                                 8.2  0.0  14    1.00 1.00                 0.0  0.0                         3.39.sup.e                              3.20.sup.e                                 7.5  9.0  15    0.98 0.92                 0.0  0.0                         1.31.sup.f                              1.24.sup.f                                 9.0  9.0  16    1.08 1.04                 0.0  0.0                         1.74.sup.e                              2.05.sup.e                                 10.0 10.5  17    1.11 1.01                 0.0  0.0                         0.89 0.96                                 0.0  0.0Alcohol  18    0.89 1.00                 0.0  0.0                         0.91 1.05                                 0.0  0.0Supernatant  19    1.08 1.07                 0.0  0.0                         1.05 0.96                                 0.0  0.0  20    1.02 1.00                 0.0  0.0                         1.00 0.94                                 0.0  0.0__________________________________________________________________________ .sup.a Test reactions 5 days before treatment with TFprecipitate .sup.b Test reactions 7 days after intraperitioneal injectin of TFporecipitate, equivalent to 400 mg unfractionated TF .sup.c E. bovis  antigen: Optimal antigen concentration for blastogenesis and 800 μg antigen for skin test .sup.d Purified protein derivative: Optimal antigen concentration for blastogenesis and 800 μg antigen for skin test. .sup.e p&lt;0.01 .sup.f 0.01 &lt;p &lt;0.1 
    
     
                                           TABLE__________________________________________________________________________TRANSFER OF CMI WITH ALCOHOL FRACTIONATED TF.sub.d      Before TF.sub.d.sup.a              After TF.sub.d.sup.b                      Skin reaction                              Skin reaction                      7 days after                              10 days after     Blastogenesis:              Blastogenesis:                      treatment:                              treatment:     stimulation              stimulation                      diameter of in-                              diameter of in-     index    index   duration (mm)                              duration (mm)Treatment Animal      E. bovis          PPD.sup.c               E. bovis                   PPD                      E. bovis                           PPD                              E. bovis                                   PPD__________________________________________________________________________Alcohol 21  1.03 1.09              1.62.sup.d                   1.19.sup.f                      0.0  0.0                              10.0 8.0PrecipitatesCombined 22  0.96 1.09              1.44.sup.d                   1.16.sup.f                      0.0  0.0                              12.0 10.0 23  1.00 1.00              1.27.sup.e                   1.84.sup.d                      0.0  0.0                              9.0  8.9 24  0.94 0.98              1.74.sup.d                   1.40.sup.d                      0.0  0.0                              10.5 9.1Second 25  1.04 1.01              2.80.sup.d                   3.20.sup.d                      12.0 12.4                              ND.sup.g                                   NDAlcoholPrecipitate 26  1.10 0.93              2.08.sup.d                   2.25.sup.d                      18.5 15.0                              ND   ND__________________________________________________________________________ .sup.a Test reactions 5 days before treatment with TF precipitate, equivalent to 400 mg unfractionated TF .sup.b Test reactions 7 to 10 days after treatment .sup.c Purified protein derivative and E. bovis  antigen: Optimal antigen concentration for blastogenesis and 800 μg antigen for skin test. .sup.d p &lt;0.01 .sup.e 0.01 &lt;p &lt;0.1 .sup.f Not significant .sup.g ND; not done