WO1983000017A1

WO1983000017A1 - Anaplasma antigen

Info

Publication number: WO1983000017A1
Application number: PCT/US1982/000816
Authority: WO
Inventors: Of Illinois Foundation University; Miodrag Ristic; Michael G. Levy
Original assignee: Univ Illinois
Priority date: 1981-06-22
Filing date: 1982-06-17
Publication date: 1983-01-06
Also published as: EP0081575A4; EP0081575A1

Abstract

Water soluble antigens specific for Anaplasma sp. are produced and isolated in vitro. For example, a non-living, stable, soluble Anaplasma marginale antigen (designated "Anablock") is characterized, interalia, as a glycoprotein having a molecular weight of about 53,000 and an isoelectric point of about 6. Vaccine compositions providing immunologically effective quantities of soluble specific antigen and a suitable adjuvant are capable of provoking protective immune response to challenge with virulent Anaplasma, e.g., blocking the development of virulent A. marginale infection. The protective immune response in vaccinates is serologically characterized by positive results in latex agglutination (LAT) and fluorescent antibody (IFA) test, but negative results in complement fixation (CF) and capillary tube agglutination (CA) test.

Description

ANAPLASMA ANTIGEN

BACKGROUND

The present invention relates generally to materials and methods useful in immunization of animals against infection by parasites of the genus Anaplasma (Order Rickettsiales, family Anaplasmatacae). More specifically, the present invention provides novel, in vitro produced and isolated, water soluble antigen materials specific for Anaplasma __., which, when administered as an active component of a vaccine, provoke develop¬ ment of an in vivo protective immune response in the vaccinates as well as a characteristic serologic profile in in vitro tests.

Anaplasmosis is a tick-borne hemotropic disease of cattle and certain other ruminants. Its causative agents include the three recognized species of Anaplasma, i.e., A. marginale, A. eentrale, and A. ovis. Occa¬ sionally, anaplasmosis has been noted to involve a "mixed" infection of A. marginale and related Par anaplasma species, __ caudatum and/or P. dis- coides. Bovine anaplasmosis is manifested by progressive severe anemia associated with the presence of parasite "inclusion bodies" in erythrocytes. The disease is quite frequently fatal, especially among cattle initially infected as adults.

Infection with A. marginale is widespread among cattle popu¬ lations of the world and especially throughout tropical and subtropical geographic regions, causing millions of dollars of damage annually. Rumi¬ nants other than cattle found to be susceptible to A. marginale infection include the zebu, water buffalo, gnu, blesbuck, duiker, wildebeast, African antelope, American deer, elk, bison and cameL Sheep and goats have been observed to develop transient, subelinical A. marginale infections.

A. eentrale also has cattle as its principal host but is rela¬ tively less virulent than A. marginale. Anaplasmosis involving A. eentrale is frequently distinguished from the disease state involving A. marginale by the location of the parasite within infected erythrocytes. A. ovis has sheep as its principal host but also infects goats and deer, causing mild to severe anaplasmosis symptoms. Numerous prior publications by co-inventor Ristic and his co- workers have provided a comprehensive review of current knowledge con¬ cerning the anaplasmosis disease state, its causative agents and available therapeutic and immunoprophylactic methods and materials for its preven¬ tion and treatment. See, e.g., Ristic, et aL, Anaplasmosis in "Immunity to Blood Parasites in Animals and Man", pp. 151-188, Plenum Press, New York, N.Y. (1977) and Ristic, Anaplasmosis, in "Bovine Medicine and Surgery", VoL I, pp. 324-348, American Veterinary Publications, Santa Barbara, Ca (1980) which are expressly incorporated by reference herein for the purpose of providing in ormation as to the background of the invention.

A number of therapeutic agents have been found to be moderately effective in treating anaplasmosis in cattle. As one example, Anaplasma sp. parasites are sensitive to treatment with broad-spectrum antibiotics such as the tetraeyclines. Due to a characteristic long delay between initial infection and onset of clinical symptoms of anaplasmosis, however, tetraeycline treatment is seldom initiated until late in the acute disease phase when it is generally least effective. Because of the lack of effectiveness of therapeutic measures, significant effort has been directed to immunoprophylactic measures for control of the disease.

A variety of serologically demonstrable antibodies appear in the course of anaplasmal infection and detection of these antibodies by various serodiagnostie methods has been important in field diagnosis and control of anaplasmosis. None of the isolated antibodies, however, have been found to be useful in transfer of passive immunity to anaplasmosis.

Among the earliest of attempts at ϊmmunoprophylaxis was a "premunition" procedure involving purposeful infection of an animal (with, e.g., whole blood of an A. marginale infected animal) followed by treatment with antibiotics in the hope of lessening clinical signs of the disease. Expectedly, premunition has proven to be too hazardous, costly and time- consuming to warrant widespread application.

Several vaccination techniques have b'eeπ proposed to be useful in immunoprophylactic control of A. marginale infection. A first proposed vaccination technique involves administering less virulent A. eentrale parasites for the purpose of developing a cross-protective immune response to A. marginale. A second proposed technique involves use of an "inactivated" or "killed" immunogen (consisting of marginal bodies and erythrocyte ghosts) derived from the blood of acutely infected cattle. See, e.g., U-S. Letters Patent No. 3,511,908. In a third proposed technique, the vaccine comprises a live, attenuated form of A. marginale selected by gradual adaptation of the organism to growth in an atypical host, the sheep. None of the above-noted vaccination techniques has been put to widespread use. The live A. eentrale vaccine has been found to display quite unpredictable virulence; the inactivated A. marginale vaccine has shown low efficacy and induction of isoimmunity to blood groups as evidenced by, e.g., erythroblastosis in calves of immunized cattle; and the live attenuated A. marginale vaccine is generally unsuitable for field use due to cold storage requirements. Significantly, animals vaccinated accord¬ ing to any of the above techniques cannot be distinguished serologically from actively diseased or recovered animals.

Compounding the search^' for non-virulent Anaplasma immuno- gens suitable for use as vaccine components is the antigenic complexity of anaplasmal species. The known antigenic profile of Anaplasma includes both intraerythrocytic and serum "antigens" of widely varying constitution and properties.

A number of researchers have isolated soluble and insoluble "antigens" from lysed infected erythrocytes, some of which are used in diagnostic tests. The active component of the well-known complement fixation test (CF) is an intraerythrocytic lipoprotein. See, Gates, et aL, Proc. A.V.M.A., 91st Ann. Mtg. (1954). The particulate "antigen" of the capillary tube agglutination (CA) test has been shown to consist of struc¬ tures closely resembling Anaplasma initial bodies. See, Ristic, J.A.Y.M.A., Ml, p. 588 (1962). None of the intraerythrocytic "antigens" has been useful in eliciting a protective response when administered as a vaccine compo¬ nent.

Soluble isolates obtained from serum of infected animals have displayed lipoprotein or glycoprotein electrophoretic mobility. Vaccines containing certain of these soluble serum "antigens" are capable of eliciting in vivo production of precipitating antibodies. None, however, is capable of

_ provoking an immune response protective against death of vaccinates subjected to virulent challenge with A^ marginale.

In the hope of obtaining large quantities of parasites for study and as a potential source of antigenic materials, substantial research effort has been directed toward developing methods and materials for in vitro cultivation of Anaplasma. See, e.g., U.S. Letters Patent No. 3,616,202. These efforts have met with marginal success until very recently when some degree of continuity in cultivation and parasite propagation was achieved by Kessler, et aL, Am. Jour. Vet. Res., 40, pp. 1767-73 and 1777-80 (1979) through modification of the Tt-ager, et aL methodology proposed for Plasmodium production. [See, Trager, et aL, Science, 193, pp. 673-5 (1976) and Jensen, et aL, J. ParasitoL, _Z, pp. 883-6 (1976).] Also found to be useful in the in vitro propagation of Anaplasma sp. are the macroaerophilous stationary phase ("MASP") propagation techniques of the co-inventors which are described in their co-pending U.S. patent application Serial No. 130,482, filed March 31, 1980. See also, Levy and Ristic, Science, 207, p. 1218 (1980).

The above brief background statement reveals a long-standing need for improved materials useful as vaccine components in immunopro¬ phylactic treatment of cattle and other ruminants prey to anaplasmosis. A suitable anaplasmal immunogen should have the following characteristics which are not presently possessed by any single immunogen available to the art. It should be susceptible to production in vitro, preferably by means of generally continuous cultivation of parasites. It should have relatively distinct physical properties so that it can be isolated with- ease. It should be water soluble, thereby allowing for uniform formulation in a vaccine and for admixture with soluble drugs and other antigens used in vaccination against other diseases. Both the immunogen and vaccines prepared therefrom should be storage stable for long periods of time without the need for costly or difficult maintenance procedures. The immunogen should be non-virulent and should not induce iso-blood group immime responses, yet it should be capable of provoking a long-term, anemnestic, protective response to challenge of virulent anaplasmal infection. Finally, the protective response developed should preferably be of a character permitting distinction of vaccinated animals from diseased or recovered animals.

-- KE

OMP BRIEF DESCRIPTION

The present invention comprehends novel Anaplasma antigen preparations, vaccines including i munologically effective amounts of the same as an active immunogen, and methods for immunoprophylactic treat¬ ment of animals susceptible to anaplasmal infection with such vaccines. More specifically, the invention provides new, in vitro produced and isolated, water soluble antigen substances specific for Anaplasma sp., especially A_ marginale. Presently preferred methods for in vitro preparation of a specific A. marginale antigen (designated "Anablock") involve parasite propagation in erythrocyte host cells according to hereinafter specified modifications of published cultivation techniques, followed by isolation of the soluble antigen from the medium supporting culture growth. Anablock antigen so prepared and isolated is characterized by: solubility in water and normal saline solution; insolubility in from about 40 to about 75% ammonium sulfate solution; the presence of carbohydrate and protein components (as well as the absence of any functionally essential lipid component); a molecular weight oi the order of about 53,000; an isoelectrie point of about 6.0; heat stability at 56° C for at least 30 minutes; and immunodiffusion test reactivity with antibodies from animals recovered from virulent A. mar¬ ginale infection.

Vaccine preparations comprising immunologically effective quantities of Anablock antigen of the invention combined with a suitable diluent, adjuvant and/or carrier substance are storage stable at ambient conditions over long periods of time, are non-inflammatory upon parenteral administration, and are uniquely characterized by in vivo immunological effects including the absence of virulence, the absence of manifest induc¬ tion of iso immunity and, most significantly, the provocation of a long-term anemnestic protective response to virulent infection by A. marginale.

Immunoprophylactic methods of the invention include, e.g., parenterally administering Anablock antigen-containing vaccine to develop protective immunity in vaccinates which is characterized by a serological property profile directing positive reaction in fluorescent antibody (I A) and serum latex agglutination (LAT) test procedures but negative response in complement fixation (CF) and capillary tube agglutination (CA) tests. This serologic profile is easily distinguished from infected or recovered animals which display positive responses in CF and CA results.

Other aspects and advantages of the invention will become apparent upon consideration of the following detailed description.

DETAILED DESCRIPTION

The following illustrative examples relate to presently pre- ferred methods of practice of the invention. More specifically, the examples describe: a method for in vitro propagation of A. marginale which gives rise to a specific Anablock antigen-enriched growth medium; an alternate in vitro propagative method; methods for isolation of Anablock from culture growth medium; physical and biological characteristics of Anablock antigen; methods for formulating vaceine preparations; and, in vivo tests of the efficacy of vaccine preparations including Anablock antigen.

EXAMPLE 1

An A. marginale enriched growth medium from which soluble Anablock antigen of the invention can be isolated is obtained by practice of the Anaplasma propagative techniques described by Kessler, et al. Am. Jour. Vet. Res., 40, pp.1767-73 and 1777-80 (1979). Blood from cattle infected with virulent A^ marginale is collected in acid-eitrate-dextrose solution during the early stages of parasitemia. Erythrocytes are separated by centrifugation at 600 x g for 15 minutes at room temperature. After removal of plasma and buffy coat cells, the erythrocytes are resuspended and washed twice in a medium consisting of RPMI 1640 supplemented with 25 mM HEPES buffer and 10% bovine serum (?H 7.4). Cells are resuspended in a ratio of 1:8 (v/v) with culture medium, resulting in a final packed cell volume (PCV) of 8% to 9%. After distribution of 1.4 ml of the cell suspension into 35-mm petri dishes, the cultures are incubated under reduced oxygen tension in a candle jar at 38° C. The medium is changed daily. Subcultures are effected by centrifuging the primary culture and

OMPI ^ . WIPO resuspending these cells with sufficient fresh medium and normal, freshly collected bovine erythrocytes to obtain the desired dilution.

EXAMPLE 2

An antigen enriched growth medium from which a soluble antigen of the invention can be isolated is also obtained through modifica¬ tion of the macroaerophilous stationary phase propagation techniques of the co-inventors as set out in Science, 207, p. 1218 (1980). More specifically, blood is collected from a splenectomized Bos tauras calf infected with virulent A. marginale parasites and immediately defibrinated by shaking with glass beads. The defibrinated blood is sedimented at 2,000 x g for 15 minutes and the serum and buffy coat cells are removed. The remaining erythrocytes are washed two times in phosphate buffered saline (pH 6.8) and added to a medium consisting of 90 percent Medium 199 (containing 25 mm HEPES) and 10 percent heat inactivated (56° C/30min) normal bovine serum. The final content of the mixture is about 9%. This mixture (pH 7.2) is transferred to plastic culture flasks so that the final depth is approximately

0.6 em and then incubated under an atmosphere of 5% CO₂/humidified air at 37-38° C. At one or two day intervals the overlying supernatant is removed and replaced with fresh erythrocyte-free media.

While the foregoing two Examples are correlated to presently preferred m vitro propagative procedures, it is expected that more efficient propagative techniques may be developed which will allow for enhanced generation of soluble antigen. As presently understood, any in vitro propagative technique which promotes growth stage development of Ana¬ plasma initial bodies should provide for enrichment of the growth culture medium with soluble antigens of the invention. Availability of the soluble specific antigen by the procedure of Examples 1 and 2 will make possible further purification by straightforward means and the eventual determina¬ tion of whether lower molecular weight fragments of the relatively crude antigen isolates are or can be responsible for in vivo protective activity. Chemical synthesis or recombinant DNA techniques may thereafter be employed to generate in large quantities the antigen or any lower molecular weight, immunologically active fragment or fragment analog. EXAMPLE 3

Saved culture medium portions obtained according to Example

2 are pooled. The supernatant containing the soluble antigen is centrifuged at 12,000 x g for 30 minutes and passed through a 0.22 micron millipore filter. The antigen-containing filtrate may be maintained in a frozen state for long periods of time or lyophilized and stored at 4° C. under vaceum.

EXAMPLE 4

The following determinations of physical and biological char¬ acteristics of Anablock antigen of the invention were performed on a sample of antigen prepared according to Example 3.

Molecular weight analysis by sodium dodecyl sulfate-poly- acrylamide gel electrophoresis showed a molecular weight of about 53,000. The presence of a proteinaceous component was verified spectrophotometrically by absorption of ultraviolet light (280 nm) and by biuret chemical reaction.

The presence of a* carbohydrate component was verified by susceptibility to alpha-amylase degradation in the following procedure. Alpha-amylase (alpha-l,4-glucan 4-glucanohydrolase, E.C. No. 3.2.L1, Sigma Chemical, St. Louis) was dissolved in 0.02 M phosphate buffer (pH 7.0) and added to lyophylized culture supernatant according to Example 3 in a ratio of 2 mg enzyme to 100 g antigen-containing lyophilizate. The sample was then incubated at 37° C for 16 hours. Crossed immunoelectrophoretic analysis showed partial degradation of antigen when reacted with hyper- immune rabbit anti-A. marginale serum. Antigen degradation is made evident by significant reduction of an antigen-antibody precipitation reac¬ tion. This, in turn, indicated that carbohydrate moities contribute to antigenic structure which is largely proteinaceous in nature.

The antigen was found to be soluble in water and normal saline but insoluble in 40 to 75% solutions of ammonium sulfate.

Heat treatment at 56° C for 30 minutes did not result in any degree of degradation evident in immunodiffusion test reactivity with antibodies from cattle recovered from virulent A. marginale infection. Analysis by isoelectric focusing revealed an isoelectric point of about 6.

EXAMPLE 5

A vaccine is prepared in the following manner. Cultures were prepared in flasks according to Example 2 utilizing cells with an initial parasitema of 8.0 percent and a total culture volume of about 110 ml per flask containing about 9% erythrocytes. Supernatants were collected at 2 day intervals and processed according to Example 3. For each vaccine dose, twenty ml of supernatant collected at 96 hours a ter initiation of cultures is lyophilized and stored under vacuum at 4° C. This quantity of the lyophilized material (about 400 mg) was reconstituted in 1 mL of 1 ^• gram/liter aqueous solution of Saponin adjuvant (Quil-A of Superfos Export Co., Copenhagen, Denmark) to yield 1 ml of vaccine.

EXAMPLE 6

A vaccine efficacy study was conducted at the University of Illinois involving two test and two control Holstein-Fresian cows.

The two test animals were vaccinated on day 0 of the test procedure and again on day 21 with 1 ml of Anablock antigen vaccine preparation obtained according to Example 5. The four cows were chal¬ lenged with virulent A. marginale on test day 4L The challenge consisted of intramuscular administration of 1 ml of bovine blood containing virulent A. marginale organisms in 73 percent of the erythrocytes.

Cattle were periodically monitored for gross physical mani¬ festations of disease such as febrile response. Serologic tests conducted during the experiment included hematocrit and packed cell volume deter- minations as well as determination of the percent of erythrocytes displaying parasite invasion (parasitemia). Immunologically based serologic determina¬ tions included analysis in complement fixation (CF) and capillary tube agglutination (CA) tests as well as fluorescent antibody (IFA) and latex agglutination (LAT) tests. IF A titers were determined according to the procedure of

Lohr, et aL Z. Tropenmed., 24, p. 86 (1973). The LAT procedure was as follows. Polystyrene latex particles (0.8 μ m diameter, 10% solid content in an aqueous suspension, Sigma Chemical, St. Louis, Mo.) were diluted 1:5 with 0.15 M glycine buffer (pH 8.3) containing 0.2% disodium ethylene diamine tetraacetic acid, spectrophotometrically standardized (a properly standard- ized suspension of latex particles had an optical density of 0.3 at 650 nm when diluted 1:100), and filtered through Whatman No. 4 filter paper. The final 2% suspension was then stored at 4° C. The latex particles in suspension were thereafter sensitized for 15 minutes at 56° C with an equal volume of an aqueous solution of lyophilized antigen according to Example 3 which had been restored to a volume equal to 0.625 to L25 times the original culture supernatant volume with distilled water. The agglutination test was performed on glass slides marked into 2.5 cm squares. One drop of sensitized latex particles was applied to each square and one drop of undiluted serum was added, mixed, hand-rotated, and the suspension exam- ined over indirect lighting for evidence of agglutination. Differing degrees of positive reactions were observed, varying from an abundance of large definitive clumps with complete aggregation (scored "+++") to fine clumps • and a granular .appearance (scored ^π+π). Negative reactions (scored "-") showed uniform turbidity without flocculation. Table 1 below sets forth data with respect to parasitemia, hematocrit and IF A tests for the experiment.

OMP TABLE 1

•

k % Hematocrit

Maximum IFA Titer IFA Titer Maximum Reduction Following at Maximum IFA Titer % Parasitized Following Vaccination Challenge Following Challenge Erythrocytes Challenge

1

Controls 1

A — < 1:40 1:10240 30 64

B — < 1:40 1:5120 35 70

Vaccinates

C 1:1280 1:40 1:10240 0.1 12

D 1:2560 1:40 1:10240 0.1 20

Prior to the experiment, all four animals tested "negatively" in serum CA, CF, IFA and LAT tests. After vaccination, but prior to challenge, body temperatures of vaccinates remained relatively stable, as did packed cell volumes. Vaccinates rather rapidly (3-5 days post challenge) developed anti-A. marginale antibodies as measured by IFA tests, indicating a strong anemnestϊc response to infection. Control animals did not develop such antibodies until 7-11 days after challenge. Post-challenge parasitemia (as determined by microscopic examination of Giesma-stained thin blood films) was quite low in vaccinates — never exceeding about 0.1%. By about three weeks after patency, parasitemia had cleared completely in vaccin¬ ates and no antibiotic treatment was required. By about 40 days after challenge, the percent of parasitized erythrocytes in the non-vaccinated control animals was about 30 to 35%, and the packed cell volumes of control animals had dropped to such low levels that tetracycline treatment was required to prevent death.

Both of the control animals developed characteristic symp¬ toms of anaplasmosis, i.e., anemia, anorexia and high temperature, while the vaccinated animals remained clinically normal throughout the experiment. Significantly, one vaccinate and one control animal were pregnant at the time of the experiment. The vaccinated animal delivered a healthy calf at 45 days after challenge. The calf was allowed to obtain colostrum and milk from its immunized mother and exhibited a passive IFA titer of 1:20,400 for more than one month. Both the cow and calf remained free of hemolytic disease indicating lack of isoimmune response to vaccination. As a result of patent A. marginale infection, however, the pregnant control animal aborted on day 34 after challenge.

EXAMPLE 7

A second study was conducted using the soluble Anablock antigen of Example 3. Four test and four control Angus heifers were employed and vaccination was according to the procedures of Example 6. Two of the test and three of the control animals had been used as vaccinate test animals in a Babesia bovis vaccine trial and entered this experiment about four months after _ bovis challenge. No variations in serologic profile in this test were attributable to the prior B. bovis vaccination history of the heifers. The test animals were vaccinated at day 0 of the test (1 mL vaccine subcutaneously at the right side of the neck), and again on day 21 (1 ml. vaccine subcutaneously at the left side of the neck). All eight animals were challenged on test day 50 by intravenous administration of blood from a cow acutely infected with A. marginale (70% parasitemia). This type of intravenous challenge procedure generally shortens the incubation period for development of anaplasmosis by about 50% as compared to the intramus¬ cular injection procedure used in prior Example 6 which more closely reproduces the natural vector-borne challenge and incubation procedure. Results for IFA, CA and LAT tests are set out in Table IL In Table II, the following symbols are used to represent LAT test data: "-" for a negative response characterized by uniform turbity without floceulation; "+" for a weak response characterized by fine clumps, granular appearance; "++" for a moderate response characterized by dis¬ tinctly larger clumps; and "+++" for a strong response characterized by large definitive clumps with complete aggregation. In the CA test shown in the Table, "-" indicates a negative response and "+" indicates a positive response and "+" indicates aft equivocal test result. IFA and CF titers of less than 1:20 and CF titers of less than 1:5 were not considered to be significant and are reported as "0".

OMPI TABLE π

A. DAY 24 DATA

^• IFA CA CF LAT

VACCINATE #1 320 0 +

#2 320 0 -

#3 640 0 +

#4 320 0 -

CONTROL #1 0 0 -

#2 0 0 -

#3 0 0 -

#4 0 0 —

B. DAY 31 DATA

IFA CA CF LAT

VACCINATE #1 640 0 ^•_^■+

#2 320 0 -

#3 * 1280 0 +

#4 640 0 +

CONTROL #1 0 0 -

#2 0 0 -

#3 0 0 -

#4 0 0 —

C. DAY 38 DATA

IFA CA CF LAT

VACCINATE #1 1280 0 -H-

#2 640 0 -

#3 1280 0 ÷+

#4 640 0 TABLE H (Cont'd.)

CONTROL #1 0 0 #2 0 0 #3 0 0 0 0

D. DAY 45 DATA

IFA CA CF LAT VACCINATE #1 1280 + 0

#2 1280 0 #3 1280 0 ++ 1280 0 +

CONTROL #1

#2 0 — 0 *

#3 0 - 0 -

#4 * 0 — - 0 "*•

*

E. DAY 52 DATA

IFA CA CF LAT

VACCINATE #1 2560 + 40 +-H-

#2 2560 + 0 -H-

#3 2560 + 80 +++

#4 2560 + ^• 40 ++

CONTROL #1 80 - 0 -

#2 80 - 0 -

#3 40 - 0 -

#4 80 - 0 ++ TABLE π (cont'd.)

F. DAY 59 DATA

IFA CA CF LAT

VACCINATE #1 2560 + 160 +++

#2 2560 + 160 ++

#3 2560 + 160 -H-+

#4 2560 + 160 -H-

CONTROL #1 1280 + 640 +-H-

#2 1280 + 320 +++

#3 1280 + 160 ++

#4 1280 - 160 -H-

G. DAY 66 DATA

IFA CA CF LAT

VACCINATE #1 2560 + 160 -

#2 5120 + 2560 +

#3 * 5120 + 640 +

#4 5120 + 2560 -

CONTROL #1 2560 + 5120 +++

#2 2560 + 640 ++

#3 2560 + 2560 ++

#4 2560 + 5120 ++

H. DAY 73 DATA

IFA CA CF LAT

VACCINATE #1 5120 + 160 ++

#2 10240 + 1280 +

#3 10240 + 640 ++

#4 10240 + 2560 ++

- uRi

O H '^" TABLE π (cont'd.)

CONTROL #1 5120 + 640 +++

#2 5120 + 2560 ++

#3 2560 + 1280 -H-

#4 5120 + 2560 ++

I. DAY 79 DATA

IFA CA CF LAT

VACCINATE #1 5120 + 320 +++

#2 10240 + 2560 -H-+

#3 10240 + 320 -H-+

U 10240 + 160 ++

CONTROL #1 5120 + 2560 -H-+

#2 5120 + 2560 -H-

#3 5120 + 80 -H-

#4 5120 + 1280 ++

While the experimental data do not illustrate as dramatic a protective response to anaplasmosis as was noted in Example 6, all measured parameters of severity of infection indicated significant advantages in response to infectious challenge for the vaccinated test animals over the controls. Because none of the animals died as a result of challenge, one of the most significant distinctions between test and control animals was the difference in average lowest packed cell volume. The vaccinates displayed lowest PCV's of 22 percent while that of the controls was 15 percent.

In each of the test procedures of Examples 6 and 7, the immunoprophylactic methods were characterized not only by provocation of protective immune responses but by distinct serological profiles. Serum of vaccinated animals tested positively in LAT and EFA tests but negatively in CF and CA tests. Following challenge both vaccinates and controls became positive in the CF and CA tests. This distinction in serologic test results between vaccinated but uninfected animals and actively infected animals is of great practical importance in development of control programs for anaplasmosis.

While the vaccine dosages employed in Examples 6 and 7 provided a total dose of about 40 mg. of the relatively crude Anablock antigen of Example 3 per 100 pounds of body weight, it will be understood that immunoprophylaxis may be provoked by vaccines providing from about 0.1 to about 1000 mg. of antigen per 100 pounds of body weight. Similarly, while subcutaneous administration of vaccines is presently preferred other parenteral modes of administration (intravenous, intramuscular, etc.) are within the contemplation of the invention and may involve use of adjuvants other than saponin. Oral administration is also contemplated, provided that the antigen is capable of surviving digestive conditions (with or without enteric coating) and is absorbable in immunologically active form through digestive tract tissue. Due to the solubility and storage stability characteristics of the Anablock, A. marginale antigen materials of the present invention, it is expected that vaccines may be prepared which contain the antigen in combination with other therapeutically and immunologically active water soluble substances^* such as drugs and antigens protective against other diseases.

While the above illustrative examples are specifically directed to A. marginale antigen, vaccines and immunoprophylactic methods, the invention will be understood to comprehend A. eentrale and A. ovis materials and methods. Numerous modifications and variations of the above-described invention are expected to occur to those skilled in the art and consequently only such limitations as appear in the appended claims should be placed thereon.

OMP

Claims

WHAT S CLAIMED IS:

1. In vitro produced and isolated water soluble antigen specific or Anaplasma sp. parasites.

2. An antigen of claim 1 which is Anablock antigen, character¬ ized by being specific for A. marginale parasites.

3. An antigen according to claim 2 and further characterized by having carbohydrate and protein components, a molecular weight of about 53,000 and an isoelectric point of about 6.

4. An antigen according to claim 2 and further characterized by the in vivo capacity to provoke an anemnestic protective response in an animal subject to virulent infection by A. marginale.

5. An antigen according to claim 2 and further characterized by in vivo non-virulence and inability to invoke an iso-blood group immune response in an animal subject to virulent in ection by A_ marginale.

6. A vaccine composition comprising: an in vitro produced and isolated water soluble antigen speci¬ fic for Anaplasma sp. parasites; and, an immunologically acceptable diluent, adjuvant or carrier.

7. A vaccine according to claim 6 wherein said antigen is Anablock antigen, characterized by being specific for A. marginale para¬ sites.

8. An immunoprophylactic method comprising administering to an animal susceptible to infection by Anaplasma parasites an immunologi¬ cally effective amount of a vaccine according to claim 6 or claim 7 whereby the recipient animal develops an anemnestic protective response to virulent anaplasmal infection and serological characteristics distinct from those of an animal actively or previously infected by Anaplasma parasites.

9. Water soluble Anablock antigen produced in a culture medium sustaining in vitro cultured growth of Anaplasma marginale para¬ sites and isolated from said medium.

10. A vaccine composition comprising: Anablock antigen according to claim 9; and, an immunologically acceptable diluent, adjuvant or carrier.

-• TE

OMPI