RICKETTSIAL ANTIGENS FOR VACCINATION AND DIAGNOSIS

Abstract: Purified antigenic surface proteins of Anaplasma marginale have been identified, and are capable of inducing immune responses in ruminants which neutralizes virulent Anaplasma marginale. The antigenic surface proteins have one or more components having electrophoretic mobilities corresponding to a molecular weight of about 15,000 daltons, 86,000 daltons, 61,000 daltons, 36,000 daltons, 31,000 daltons, or 15,000 daltons, and can be purified by an immunoaffinity chromatography process comprising the steps of disrupting Anaplasma marginale initial bodies by treatment with a detergent, passing the disrupted initial bodies over a chromatography column comprising an insoluble matrix coupled to monoclonal antibodies against a determinant on said antigenic surface protein to selectively bind said antigenic surface protein to said monoclonal antibodies and recovering the bound substantially pure antigenic surface protein from said insoluble matrix. The antigens have further utility in diagnostic tests for anaplasmosis. They can be synthesized by polypeptide procedures or by genetic engineering. DNA and amino acid sequences have been developed for at least some of the antigens according to this invention. The antigens may be useful for rickettsial organisms other than Anaplasma marginale.

Description: RICKETTSIAL ANTIGENS FOR VACCINATK-N AND DIAGNOSIS CROSS REFERENCES TO RELATED APPLICATIONS This is a continuation-in-part of application Serial No. 253,143 filed October 4, 1988. This is also a continuation-in-part of applicatio Serial No. 141,505, filed January 7, 1988; which was a continuation application Serial No. 761,178, filed July 31, 1985 (now abandoned which in turn was a continuation-in-part of application Seri No. 715,528, filed March 25, 1985 (now abandoned). This is furth a continuation-in-part of application Serial No. 245,855, filed Septemb 16, 1988. FIELD OF THE INVENTION The present invention primarily relates to antigenic polypeptid and proteins, related vaccines and methods useful to induce an immu response which is protective to reduce the severity or prevent infectio by rickettsial parasites of the order Rickettsiales, family Rickettsia, mo particularly rickettsiae (or rickettsias) of the genus Anaplasma, even mo particularly rickettsias of the species Anaplasma marginale. BACKGROUND OF THE INVENTION Rickettsiae are very small parasitic microorganisms (approximat 0.2 micron) which are of the taxonomical order Rickettsiales, fam Rickettsia. Rickettsial diseases caused by these parasites have been ve significant throughout history to both humans and animals. Hum deaths caused by outbreaks of epidemic typhus and scrub typhus num l in the millions. Epidemic typhus is caused by the rickettsia _. Rickettsia prowazelά. Scrub typhus is caused by the rickettsia 3 Rickettsia tsutsugamushi which is stϋl endemic in many rural areas of 4 Southeast Asia and Japan. Rocky Mountain spotted fever, caused by 5 Rickettsia rickettsu, is widespread in the eastern United States and is a 6 risk in many other parts of the country. 7 Animal diseases caused by rickettsiae include Rocky Mountain 8 spotted fever and canine ehrlichiosis, caused by Ehrlichia canis, both of 9 which afflict dogs. Rickettsial diseases of horses include equine 10 ehrlichiosis, caused by Ehrlichia equis, and Potomac fever, caused by u Ehrlichia risticu. Serious losses occur to cattle from the rickettsia 12 Anaplasma marginale. Some animal rickettsial diseases are communicable 13 to humans, for example, Q-fever, canine ehrlichiosis and Potomac fever. 14 Despite the widespread significance of rickettsial diseases, little has been is known about the molecular biology of the rickettsiae. 16 Aπaplasmosis is an arthropod borne hemoparasitic disease of cattle 17 and other ruminants caused by Anaplasma marginale. Anaplasmosis 18 occurs worldwide and severely constrains livestock production in tropical 19 and subtropical regions. This rickettsia is transmitted by ticks, biting 20 flies, and blood contaminated fomites to susceptible animals, where it 21 infects red blood cells (erythrocytes). Anaplasma marginale occurs in 22 the red blood cells as an intraerythrocytic initial body, which is a single Anaplasma marginale organism in a mature infective stage of the microbe's life cycle. The infective initial bodies reproduce by binary fission within the erythrocytes to form two to eight initial bodies which are subsequently released to infect additional erythrocytes. During acute infection the level of these parasites increases geometrically and severe cxtravascular anemia occurs. Marked weight loss, abortion, and death can occur during the acute crisis caused by this parasitic infection and the resultant parasitemia. Animals that recover from the acute infection remain persistently infected and are a reservoir for transmission to susceptible animals. Current immunoprophylaxis for anaplasmosis includes premunization with a less virulent Anaplasma marginale isolate or Anaplasma centrale, a less virulent anaplasma species. Premunization is typically followed by tetracycline treatment to control severe infection in some animals. Another immunoprophylatic approach is vaccination with a vaccine containing failed whole Anaplasma marginale organisms and host erythrocyte stroma. Premuπition is successful in controlling severe clinical disease when cattle are challenged with a virulent isolate. However, clinical disease including weight loss, abortion and occasionally death may result from premunizing inoculum. This inoculum may also transmit other hemoparasites, such as Babesia, Theileria, and Trypanosoma, and viruses, such as leukemia virus, to the animal being treated. Challenge of cattle immunized with the killed Anaplasma marginale-eτyt τocyte stroma vaccine results in mild clinical disease and persistent infection. In addition, the presence of eiythrocyte stroma in the vaccine has been shown to induce anti-eiythrocyte antibodies which can be transferred through a ' cow's colostrum to a nursing calf thus causing the autoimmune disease neonatal isoeiythrolysis. Accordingly, there remains a strong need for improved immunization techniques effective against these and other rickettsial diseases. There also remains a continuing need for relatively simple diagnostic tests for detecting carriers of rickettsial parasites. BRIEF DESCRIPTION OF THE DRAWINGS Drawings showing Figs, which relate to preferred embodiments of this invention are included herewith, and are briefly described as set forth below. Fig. IA is a reproduction of four radiographs (l)-(4) showing the detection of native Anaplasma marginale proteins on nitrocellulose using four different types of antibody or antiserum. Fig. IB is a reproduction of a radiograph showing the detection of proteins from recombinant-plasmid-containing E. coli on nitrocellulose. The p oteins where screened for reaction with rabbit antiserum R873, which is reactive to the native Anaplasma marginale surface protein complex alternatively referred to as MSP-1 or Aml05. Fig. 2 is a restriction enzyme map showing relevant portions of the Anaplasma marginale gene coding for the expression of the protein recombinant Aml05. The relative orientation and relationship of the gene as incorporated into the recombinant plas ids pAM22, pAM25, pAM97, and pAM113 are also shown. Fig. 3 is a reproduction of a radiograph showing electrophoretically separated Anaplasma marginale proteins, proteins from recombinant E coli having plasmid pAM25, proteins from E coli with plasmid pBR322, and molecular weight standard proteins. Fig. 4A is a reproduction of a radiograph showing electrophoretically separated proteins including recombinant Aml05, native Aml05 (including Aml05L and Aml05U), E. coli cells containing recombinant plasmid pAM25, and E. coli cells containing plasmid pBR322. Fig. 4B is a reproduction of a radiograph showing electrophoretically separated polypeptide fragments resulting from treatment of recombinant Aml05, and purified native proteins Aml05L and Aml05U after treatment with a protease. Fig. 5 is a reproduction of a radiograph showing electrophoretically separated proteins including recombinant Aml05, native Aml05L, and native Aml05U after immunoprecipitatioπ with monoclonal antibodies IE, and 22B 15 and rabbit antisera R911 and R907. 1 Fig. 6 is a reproduction of a radiograph showing electrophoretically 2 separated proteins resulting from the surface radiolabeling and 3 immunoprecip-tation of Anaplasma marginale initial bodies using 4 monoclonal antibodies IE, and 22Bj, and rabbit antisera R911 and R907. 5 Fig. 7A is a reproduction of a radiograph showing 6 electrophoretically separated DNA comparing Anaplasma marginale genomic 7 DNA versus recombinant plasmid DNA using Southern blotting. * Fig. 7B is a reproduction of a radiograph showing 9 electrophoretically separated DNA comparing Anaplasma marginale genomic 10 DNA versus bovine leukocyte DNA after treatment by restriction u enzymes. 12 Fig. 8 shows four restriction enzyme maps (l)-(4) for four 13 different geographical isolates of Anaplasma marginale indicating relevant 14 portions of the genome containing the gene which codes for the 15 expression of the proteins corresponding to Aml05U in the Florida 16 isolate. The identified gene areas in each map are indicated with the 17 cross-hatched bars. Below the restriction maps are five plasmid 18 diagrams indicating in heavy line the portion of the plasmids which 19 incorporated part or all of the indicated genes. The portion of the 20 plasm " d not incorporating the recombinant gene DNA is shown in a 21 light line. Fig. 9 is a reproduction of a radiograph showing electrophoretically separated proteins expressed by the recombinant E. coli cell lines which incorporated the recombinant plasmids pVAl, pWAOl, pID6, and pFLlO. Also shown are native proteins from the corresponding Anaplasma marginale isolates. Fig. 10 is a sequence diagram showing DNA nucleotide sequences for the Florida, Virginia, Idaho and Washington isolates of Anaplasma marginale, including the genes which code for the expression of the MSP-la or Aml05U protein. Fig. 11A is a reproduction of a radiograph showing DNA fragment electrophoretic separations indicating the start of transcription in the Florida isolate MSP-la gene. Fig. 11B is a sequence diagram showing portions of the DNA nucleotide sequences shown in Fig. 10 for the promoter regions of the four geographical isolates of Anaplasma marginale and E. coli. Fig. 12 is a sequence diagram showing amino acid sequences for the MSP-la (Aml05U) proteins expressed by the four different geographical isolates of Anaplasma marginale. Fig. 13 is a sequence diagram comparing portions of the amino acid sequences shown in Fig. 12. The sequences shown in Fig. 13 indicate repeat patterns of five different types labeled A-E. The number of times that a particular repeat pattern is included in the protein is indicated in the chart shown at the right of Fig. 13. Fig. 14 shows a number of synthesized polypeptide sequences and whether such sequences in vitro reacted with monoclonal antibody 22B Fig. 15 is a restriction enzyme map showing the cut sites for a number of restriction enzymes upon the DNA nucleotide sequence containing the gene coding for the expression of the Anaplasma marginale protein Aml05L for the Florida isolate. Fig. 16 is a sequence diagram showing the DNA nucleotide sequence for the gene coding for the expression of the Anaplasma marginale protein Aml05L. Also shown is the corresponding amino acid sequence of protein Aml05L of the Florida isolate. SUMMARY OF THE INVENTION The present invention seeks to overcome some of the limitations of the prior art by providing improved antigens and immunogens for detecting and immunizing relative to rickettsial parasites, in particular Anaplasma marginale. The invention includes suitable purified antigens which are bound by serum antibodies, and which are in at least some cases immunogenic to reduce the severity or prevent infection by Anaplasma marginale and other rickettsial organisms having epitopes o f the same or sufficiently similar nature. The invention also includes certain monoclonal antibodies which can selectively bind antigenic components of rickettsias such as Anaplasma marginale, and provide detection and other valuable screening and diagnostic uses. Selected native proteins can be isolated from Anaplasma marginale organizisms and purified or treated to produce one or more purified immunogenic polypeptides or proteins. The invention includes the discovery that at least one native antigen having surface-exposed epitopes is common to numerous geographical isolates of Anaplasma marginale in forms which share conserved polypeptide sequences. This protein complex has been identified and purified, and is alternatively referred to a major surface protein 1 (MSP-1) and Aml05. The two component proteins of this complex are referred to as Aml05U and Aml05L. In at least one of the geographically distinct isolates of Anaplasma marginale these complexed proteins have electrophoretic mobilities which correspond to approximate molecular weights of about 105,000 daltons. In other isolates the electrophoretic mobilities and apparent molecular weights of these two complexed proteins vary, particularly with respect to one of the two complexed proteins. Other antigenic proteins have been identified from Anaplasma marginale organisms and are characterized by electrophoretic mobilities which correspond to apparent molecular weights of about 86,000 dalto__s (Am86); 61,000 daltons (Amόl); 36,000 daltons (Am36); and 15,000 daltons (Aml5). Still other antigenic proteins as identified herein are also of use in this invention. In addition to the native proteins isolated and purified from Anaplasma marginale, the antigens and immunogens according to this invention can comprise active agents formed of one or more such proteins, polypeptide fragments of such proteins, or one or more immunologically similar proteins or polypeptides produced by polypeptide synthesis or genetic engineering. Several forms of novel antigens of this invention have been produced by recombinant DNA techniques coding for the expression of recombinant antigens which have demonstrated immunogenic effect Amino acid sequences have been identified which characterize at least some of the effective antigens and immunogens of this invention. Antigenic proteins of the invention are in part purified by removing or isolating Anaplasma marginale initial bodies from cellular components of the infected erythrocytes. The significantly purified initial bodies are thereafter disrupted, such as by using a suitable detergent or by other means. Desired antigens can be purified from the disrupted Anaplasma marginale organisms, such as by passing the disrupted initial bodies over antibodies which selectively bind the desired antigens. Such can be accomplished by passing an aqueous mixture containing the disrupted initial bodies over or through an insoluble matrix, such as an affinity chromatography column. The insoluble matrix has monoclonal antibodies specific to the desired antigenic protein or peptide which recognize one or more epitopes thereon to adsorb it onto the insoluble matrix. The adsorbed antigens are further purified by washing the non-adsorbed materials of the aqueous initial body mixture through the affinity chromatography column to leave the adsorbed antigens bound to the matrix. The adsorbed antigens are recovered from the matrix to provide purified antigens according to this invention. The novel monoclonal antibodies preferably used to prepare the purified antigens, are advantageously prepared by vaccinating or otherwise inoculating mice with the appropriate rickettsial parasites, such as by injecting the mice with bovine erythrocytes infected with Anaplasma marginale. Lymphocytes are taken from the spleen of the infected mice. The lymphocytes from the mice are fused with immortal cells, such as myeloma cells, to produce hybridoma cells which are cloned to develop hybridoma cell lines. Some of the hybridoma cell lines produce monoclonal antibodies which will selectively bind to the desired antigens. The collection of hybridoma cell lines are then screened using a novel approach to identify the hybridomas of interest. The screening of the hybridomas can advantageously initially include procedures for detecting the hybridomas which produce antibodies which bind to Anaplasma marginale. This is advantageously accomplished by indirect immunofluorescence on smears of Anaplasma marginale-infected blood. The hybridomas are then further screened to determine those which produce monoclonal antibodies against specific Anaplasma marginale proteins, such as by immunoprecipitatioπ of selected proteins of Anaplasma marginale by the cell line supernatants containing the monoclonal antibodies. In particular are selected those hybridomas producing antibodies which selectively bind Anaplasma marginale proteins having surface-exposed epitopes, more particularly epitopes also bound by immune serum of an animal previously infected by the parasites. Additional amounts of the desired monoclonal antibodies are advantageously produced by collection of ascitic fluid from mice inoculated with the selected hybridoma cell lines. Such a monoclonal antibody collected from murine ascitic fluid is appropriately purified, such as by precipitation, dialysis and chromatography. The purified monoclonal antibody is then coupled to an insoluble matrix such as Sepharose to prepare an immunoaffinity matrix. Partially purified disrupted rickettsial organisms, such as Anaplasma marginale initial bodies, are then passed through the immunoaffinity matrix and the desired antigenic protein is selectively adsorbed onto the matrix. The purified protein which has adsorbed ont-._ the matrix is then appropriately removed or otherwise recovered from the matrix to provide significant amounts of the desired antigen in a sufficiently purified form to serve effectively in the indicated uses for this invention. The degree of purity of the proteins achieved in accordance with the present invention is dependent upon the method of production used. The purity of native proteins and polypeptides derived therefrom is significantly higher than the purity of the antigen in its natural state. As an example, in its natural state Aml05 is believed to be present in an amount of about 0.1 to 1% of the total protein present in the initial bodies. In its natural state, many other impurities such as about 200 other proteins, carbohydrates, red cells, glycoproteins, and nucleic acid are present. However, the Aml05 can be purified to significantly higher levels of purity using the methods taught herein. Purity levels of approximately 10% by weight or higher are believed operable. Purity levels of 20% by weight or higher are more preferred. Still more preferred are purity levels of 50% by weight or higher. The purification techniques taught herein are capable of producing purity of at least 90 weight percent, preferably at least 95 weight percent and most preferably at least 98 weight percent. The purified Am 105 has a molecular weight of about 105,000 daltons as measured by electrophoretic mobility analysis but significantlv les when molecular weight is determined by DNA and amino acid sequence information as presented herein. Other antigens according to this invention are expected to also show significant differences between molecular weight measured by electrophoretic mobility versus sequenced information. Nonetheless the electrophoretic mobility information provides a valid means for identifying and isolating the antigens according to this invention. One of the demonstrated immunogenic antigens of this invention, Aml05 (Florida isolate), is reactive with monoclonal antibodies produced by hybridomas cell lines ANA 15D2 and ANA 22B1. Deposits of cell lines ANA 15D2 and ANA 22B1 have been made in the American Type Culture Collection under ATCC Nos. HB9046 and HB9047, respectively. The immunoaffinity purified antigens of this invention such as Aml05, and the recombinant or artificially synthesized peptides as taught herein are most preferably substantially free of contaminating glycoproteins, carbohydrates, red cells, and nucleic acids. Active fragments or subunits of the identified antigenic polypeptides of this invention may be effective in inducing immunity to Anaplasma marginale or other rickettsial organisms. Effectiveness of at least some of the antigens has been demonstrated in cattle. The size of the active fragment(s) may be as small as six to twenty or possibly s'x to ten amino acids. The antigens according to this invention can be produced by immunoaffinity chromatography as described above and elsewhere herein, or using artificial methods of polypeptide synthesis, or using genetic engineering with expression of the desired peptide(s) or protein(s). Various methods for producing polypeptides by artificial synthesis are known in the art and will not be described herein because of the well- known effectiveness of such methods in generating polypeptides with a known amino acid sequence. Reference can be made to commercial services and many scientific writings for examples of one of many suitable techniques for such synthesis. Since such techniques require knowledge of the desired amino acid sequence(s) of the polypeptide sought, the novel teachings contained herein will enable a variety of different synthetic antigens and immunogens to be produced for use in this invention. The invention further includes certain novel genetically engineered DNA and RNA sequences and microorganisms incorporating such sequences which have been produced for the purposes of analyzing and expressing the novel antigens according to this invention. Such recombinant microorganisms were advantageously produced by creating a pseudo-random genomic library of recombinant bacteria, such as E. coli, which incorporate novel recombinant DNA plasmids. The recombinant plasmids incorporate DNA fragments from the genome of an appropriate rickettsial parasite, such as Anaplasma marginale. The recombinant DNA plasmids were created by cleaving plasmid DNA with a suitable restriction enzyme. Similarly, Anaplasma marginale DNA is cleaved with suitable restriction enzymes to produce a large number of various Anaplasma marginale DNA fragments. The DNA strands from the plasmids and Anaplasma marginale were mixed to join fragments of each and then ligated to form recombinant plasmid vectors. The recombinant plasmid vectors were implanted into suitable hosts, E. coli, and cloned to produce a collection of recombinant bacterial cell lines. The resulting recombinant cell lines were screened for the expression of desired antigens, such as by using selected monoclonal antibodies against the parasites, as described above and elsewhere herein. Alternatively, viruses such as vaccinia virus can be used to produce recombinant viral vectors bearing nucleic acid sequences coding for the expression of the desired antigens. Recombinant RNA can alternatively, be produced to code for the expression of the desired antigens. Recombinant bacterial cell lines were developed which express antigenic recombinant proteins which mimic the surface-exposed native proteins contained in the protein complex alternatively called MSP-1 and Aml05. The invention also includes the discovery that the native proteins contained in this complex are polymorphic between different geographical isolates of Anaplasma marginale varying in size and amino acid sequences. Recombinant plasmids containing Anaplasma marginale DNA were analyzed to determine the DNA sequences associated with the expression of these related polymorphic proteins. The antigens expressed by these four geographical isolates were also analyzed to determine the amino acid sequences. The antigens were found to have a hypervariable domain of variable numbers of tandemly repeated sequences at the N end of the polypeptide. The tandem repeats consisted of polypeptides of 28 or 29 amino acids. The number of repeats varied between 2 and 8 within the group of 4 different isolates analyzed. However, all of the tandem repeats in the four isolates were found to possess conserved amino acid sequences. Monoclonal antibodies which bind to surface-exposed epitopes of Anaplasma marginale and are effective at neutralizing the infectivity of such organisms also bind to at least some of the conserved epitopes contained in the tandem repeat regions of the proteins. Novel recombinant cell lines have been developed which express proteins including the tandem repeat polypeptide sequences. Such nove recombinant-produced proteins containing the conserved polypeptid sequences have been demonstrated to cause an immune response i cattle which is effective to reduce the severity or prevent acute infectio by Anaplasma marginale. Other antigens bound by selected monoclona antibodies which are reactive with Anaplasma marginale, particularly thos reactive with epitopes shown to cause neutralization of the infectivity of the parasites, are also within the scope of this invention. The immunogenic antigens according to this invention can be used in vaccines to bring about an immune response effective to reduce the severity or prevent infection by rickettsial organisms. Such antigens should be present in a single dose of the vaccine in an amount of approximately 1-1000 micrograms, preferably 5-400 micrograms, and most preferably 10-200 micrograms. A single injectable dose will usually have a volume of about 1-2 milliliters. Therefore the concentration of purified surface antigen in an injectable vaccine composition will usually be in the range of from about 1 to about 500 rrucrogr-uααs/milliliter and preferably about 5 to about 200 miCTOgrams/milliliter and most preferably 10-500 micrograms/milliliter. The antigens can advantageously be dissolved or otherwise administered with an adjuvant, such as Freund's complete and/or incomplete adjuvants. The vaccine, in addition to containing the purified antigens and optionally an adjuvant, may also contain any other suitable pharmaceutically acceptable carrier or diluent. The pharmaceutically acceptable carrier or diluent is a compound, composition or solvent which is preferably a non-toxic sterile liquid useful for administra on of the active antigens or in some cases otherwise increasing the effectiveness of the inoculation treatments. In order to immunize ruminants, preferably young animals ar inoculated with a vaccine comprising the purified antigens and a desired adjuvants and diluents. The antigens can be purified from th parasites, produced as expressed polypeptides or proteins fro recombinant cells, or produced by artificial polypeptide synthesis. Suc purified antigens are preferably added to a suitable pharmaceutical acceptable carrier or diluent, and any desired adjuvant(s). Preferabl the animals are successively inoculated with a single dose as define above at one to six week intervals, preferably two to four we intervals about two to eight times, preferably three to five times. T purified protein(s) or polypeptide(s) should be present in the vaccine an amount(s) effective to induce an immune response in the anima being treated. Such immune response is preferably sufficient to prote the vaccinated animals so that if subsequently challenged with virule rickettsias, such as Anaplasma marginale, the degree of acute infecti is substantially reduced or even prevented. Injection will usually performed intramuscularly (i.m.) or subcutaneously (s.c). The purified recombinant, synthesized, or native polypeptides a proteins defined herein also are useful in diagnostic tests to determi whether an animal is infecteύ by an applicable rickettsial parasite, su as Anaplasma marginale. Conversely, such diagnostic tests may al incorporate one or more of the monoclonal antibodies specific infection by such organisms. The diagnostic tests are advantageously immunoassays, such as one or more types of enzyme linked immunosorbent assays, such as for serologic diagnosis of anaplasmosis. Alternatively, the assays can be radioimmuπoassays or others utilizing the selective binding of the purified antigens to antibodies raised in an infected animal, or monoclonal antibodies which specifically bind antigens L associated with the particular parasitic organisms of interest. When samples from subject animals are tested using such antigens and/or antibodies, results distinguishing infected and non-infected animals are obtainable. The discovered DNA sequence information can be used to create novel nucleic acid sequences which are useful as a nucleic acid probe which can be labelled and used to detect for the presence of hybridizing DNA or RNA, to provide a diagnostic test of great sensitivity. DETAILED DESCRIPTION OF THE INVENTION The detailed description of this invention is broken into subpar to aid in the understanding of the large amount of complex materi contained herein. PART I - MONOCLONAL ANTIBODIES AND IMMUNOAFFINIT PURIFIED ANTIGENS Preparation of Monoclonal Antibodies to Anaplasma marginale- The source of antigen, mouse immunization protocol, myeloma c lines used, culture media and conditions, and cell fusion and cloning a described in Davis et al, Development of monoclonal antibodies Anaplasma marginale and preliminary studies on their application. Pro Seventh National Anaplasmosis Conf. Oct. 21-23, 1981, Mississippi Sta University Press. The procedure can be summarized as follows. Animals - Young Hereford and Holstein cattle were use Animals to be infected with Anaplasma marginale were splenectomize Two inbred strains of mice, BALB/c and B10.A(3r), were used as source of cells to make hybridomas. These and additional strai B10.A, B10.A(5R) and B10 (2R), were used as a source of thymocyt for co-culture as feeder cells with hybridoma cells. Preparation of Anaplasma marginal -infected erythrocytes Splenectomized cattle were infected with an Idaho isolate of A. Margi as described in Davis, W.C. et al, Infect. Immun. 22:597-602 (197 1 For tissue culture studies, blood was collected in heparin every two to 2 four days after infection. Blood smears were made and stained with 3 Wright's-Giemsa stain and examined for anaplasma bodies. When 4 parasite ia reached 20-50%, blood was collected, centrifuged, freed of 5 ..the buffy coat and then frozen in 30% dimethylsulfoxide (DMSO) for 6 later use. Additional blood smears were prepared and frozen at -70°C 7 until used to detect monoclonal antibodies. 8 Preparation of antigen and immunization • Two types of 9 preparations were used as a source of antigen. In the first, frozen 10 cells (50% packed cell volume) with a parasitemic of 30% were thawed u and then immediately lysed with Tris (0.01M) ammonium chloride 12 (0.87%) solution (pH 12). The lysate was centrifuged at 800 rpm for 13 30 minutes the pellet was washed 3 times in Hanks Balanced Salt 14 Solution (HBSS) and resuspended in 10 ml of HBSS. Six mice 15 [B10_A(3R)} were injected intraperitoπeally (i.p.) with 1 ml of lysate. 16 Three days before the spleens were taken for cell fusion, the mice were 17 given an intravenous booster injection of 0.2 ml of lysate. The second 18 preparation, a lysate of infected erythrocytes, was purified on a 19 Renografin density gradient (25-55%) as described in Davis, N.C. et al, 20 Infec Immun. 22:597-602 (1978). The bands containing anaplasma bodies 21 and contaminating erythrocyte stroma were collected and washed, as 22 described above, then resuspended in 10 ml of HBSS. Five BALB/c mice were injected intraperitoneally with 1 ml for primary immunization and intravenously with 0.2 ml for booster immunization as described. All mice were immunized at least three weeks before receiving a booster injection. Myeloma cell lines used to produce hybridomas • Several HAT (hypoxanthine, amiπopterin and thymidine)-sensitive, tissue-culture-adapted mouse myeloma cell lines were used as fusion partners in the production of antibody producing hybridomas. NS1, a cell line that produces, but does not secrete K light chains, Oi et al, V.T., In Selected Methods in Cellular Immunology (Eds.) B.B. Mishell and SM Shiigi, WH Freeman and Co., pp. 351-372 (1980) and SP2/0-Agl4, a cell line derived from a Nsl-BALB/c hybrid that synthesizes neither light or heavy chains, Schulman, M. et al, Nature, 276:279 (1978) were obtained from the Salk Institute. X63-Ag8.653, another cell line that does not produce light or heavy chains, Kearney, J.F. et al, J. Immunol 123:1548-1550 (1979) was provided by M. Lostrum from the Fred Hutchinson Cancer Research Center, Seattle, WA. Culture media and culture conditions • Dulbecco's Modified Eagle Medium (DMEM), containing 13% fetal calf serum (FCS), 5 X 10 " S M 2-mercaptoethanol (2ME), penicillin (100 units/ml) and streptomycin (100 g/ml), was the primary culture medium for maintaining the myeloma cell lines and the new hybridoma cell lines. Glutamine (2mM) 1 was added when the medium was used 14 days after preparation. 2 Fusion and transfer media were prepared by adding HAT and HT 3 (hypoxanthine and thymidine), respectively, according to the method of 4 Littlefield, Monoclonal Antibodies-Hybridomas: A New Dimension of 5 Biological Analysis (Eds.) Kennett, R.H. et al, Plenum Press, pp. 363- 6 416 (1981). RPMI-1640 containing 15% FCS and penicillin/streptomycin 7 was used to culture anaplasma infected erythrocytes, Davis W.C. et al, * Infect Immun. 22:597-602 (1978). All cultures were maintained in a 9 5% C0 2 gas-air mixture at 37°C lo Cell fusion and cloning - Methods for fusing spleen and myeloma u cells were similar to those described by Oi et al, supra. Myeloma cells 12 were maintained in log phase of growth in T75 flasks by removing 13 excess cells and feeding every 3 to 4 days. The day before fusing, 14 cells were collected, washed by centrifugation and plated in T75 flasks 15 at 1 x 10 7 cells per flask. On the day of use, cells were collected, 16 centrifuged and resuspended in DMEM without FCS. Mice are 17 immunized by injecting disrupted bovine erythrocytes which contain 18 Anaplasma marginale initial bodies. Spleen cells were obtained from the 19 freshly-killed, immunized mice by injecting the spleen with DMEM, gently o tearing it apart with tweezers and then pressing it through a 100 mesh 21 screen into a 50 ml test tube. Following removal of particulate debris, 22 the cells were centrifuged into a pellet and the medium removed. Contaminating mouse erythrocytes were selectively lysed by brief exposur to distilled water (2 ml) and the spleen cells were then quickly dilute in DMEM. Thymocytes to be used as feeder were collected as abov (but without water lysis) and suspended in DMEM-FCS-HAT at 1 x 10 cells per ml. Myeloma cells and spleen cells were counted, then mixe either at a ratio of 5 or 15 spleen cells to 1 myeloma cell. Usually 10* spleen cells were mixed with 2 or 4 x 10 7 myeloma cells. Th cell mixture was sedimented and the supernatant removed. One ml o a 50% solution of polyethylene glycol (PEG 1540, Baker Chemical Co. was then placed over the cell pellet and slowly mixed with the cell so as to form a slurry of small (1 mm 3 ) clumps of cells. Followin 3 minutes of mixing, the cells were slowly diluted by adding 10 m DMEM in 10 minutes and 20 ml over the next 5 minutes. Th resultant mixture of fused cells was centrifuged into a pellet and th cells were resuspended in DMEM-FCS-HAT. Thymocytes were the added to give a mixture containing 10 s spleen cells, tumor cells an 1 x 10 9 thymocytes. Cells were distributed in ten 96-well culture plate and placed in the incubator. One half the tissue culture medium wa replaced every 3 to 4 days. When clones of hybridomas were 30 1000 eel' , in size (usually by 12-18 days), the supernatants wer collected and tested by indirect immunofluorescence on smears infected erythrocytes. Cells from positive wells were identified an i transferred to 24-well (2ml) culture plates. Three to 5 x 10 6 2 thymocytes were added as feeder cells to support growth. At 14 days 3 or when the cultures needed to be thinned, the medium was replaced 4 with DMEM-FCS-HT. The cells were maintained in static cultures (1 5 week in DMEM-FCS-HT then on DMEM-FCS) for two weeks by 6 removing excess cells and feeding every 2-4 days, depending on the rate 7 of replication of individual clones. At this time, a duplicate plate was 8 prepared and allowed to overgrow. The supernatants from this plate 9 were tested for antibody activity. All cell lines identified as antibody lo producers by this procedure were then taken from the master plate and u expanded into 6 well plates (5 ml capacity in each well) as single 12 cultures, 3 wells per cell line. Cells were collected twice and frozen 13 (3-10 x 10 6 cells in 10% DMSO) in liquid nitrogen. The remaining 14 cells were allowed to proliferate and die. The supernatants were then 15 collected (approximately 15 ml) and frozen for subsequent analysis. 16 When cell lines producing antibody of immediate interest were 17 identified, they were taken from the freezer, thawed and cloned by 18 limiting dilution immediately or following 24-hrs. culture. Usually, 19 hybridoma cells were plated in 2 to 6 96-well plates, 3 cells per well 20 in the presence of 10 6 thymocytes. Wells containing single colonies 21 were identified microscopically and supernatants were collected and tested 22 for antibody activity. Cells from positive wells were transferred as above to 24-well plates and then to 6-well plates for colony expansion and preservation. Four to 6 cloned lines were preserved for each line. In preliminary studies several methods of preparing hybridomas were compared to determine the optimal conditions for use of monoclonal antibody technology in the study of Anaplasma marginale. The results obtained revealed that SP2/0- ag 14 and X63-Ag8.653 myeloma cells were the most useful fusion partners because of their growth characteristics, the efficiency of outgrowth of hybridomas, and their inability to secrete light or heavy chains. The fusion ratios found to be the most effective were 5 to 1 for SP2/0-Agl4 and 2.5 to 1 for X63-Ag8.653. Under the culture conditions employed, it was essential to have 2-ME in the culture medium and thymocytes as feeder cells. When these measures were disregarded, outgrowth of the hybridomas was unpredictable; however, when the measures described were taken, 600 to 1000 hybrids were obtained per fusion. The use of erythrocyte-contaminated preparations of Anaplasma marginale as antigen presented no problems. Both the crude lysate an renografin-purified preparations were highly immunogenic. The majorit of the hybrids detected in primary cu ' .ure produced antibody t Anaplasma marginale. More anti-erythrocyte antibody producing hybrid were seen when the lysate was used as a source of antigen, however When a sufficient number of hybridomas were collected and preserved, they were tested by immunodif usion to determine the class of antibody being produced. In addition, they were tested by indirect immimofluorescence on smears of infected erythrocytes to determine their patterns of binding to Anaplasma marginale or erythrocytes, on cultures of infected monocytes grown in short term culture and on acetone-fixed sections of tissue taken from infected cattle. Selection of Cell Lines Producing Anti-Ami 05 Monoclonal Antibodies- The hybridoma cell supernatants are initially screened for -nti-Anaplasma marginale antibody by indirect immunofluorescence on acetone-fixed smears of Anaplasma marginale infected blood. The positive (antibody producing) cell lines are then further screened for specific production of anti-Aml05 antibodies using immunoprecipitation of either [ 35 S] methionine radiolabeled or [ 12S I] surface radiolabeled Anaplasma marginale. The exact procedure for this, which is a slight modification of the procedure reported by Palmer, G.H. et al, J. Immunology, 133:1010 (1984), is as follows. The immunoprecipitation of surface-radioiodinated initial bodies and erythrocyte stroma was performed by using a modification of the technique described by Shapiro, S.Z. et al, J. Immunol. Methods, 13:153 (1976). The initial bodies or erythrocytes were disrupted with 1% (v/v) Nonidet P-40 and 0.1% (w/v) SDS at 4°C for 30 min, centrifuged at 135, 000 x G for 60 min, passed through a 0.2 m filter (Millipore Corp., Bedford, MA), and sonicated for 15 sec at 50 W. Two hundred thousand trichloroacetic acid precipitable cpm were added to 50 1 hybridoma supernatant followed by 10 microliters rabbit anti-mouse immunoglobulin in siliconized tubes and were incubated at 4°C for 30 min. One hundred microliters of 10% (v v) protein A-bearing Staphylococcus aureus (Calbiochem-Behring Corp., La Jolla, CA) were added to each tube, incubated for 30 min at 4 β C, and washed six times with Ten buffer (20 mM Tris-HCl, 5 mM EDTA, 0.1 M NaCl, 15 mM NaN 3 , pH 7.6) containing 0.1% Nonidet P-40„ and for the first four washes 2 M NaCl, by using centrifugation at 1250 X G. The precipitated label was eluted by bofling the staphylococci-bound complexes for 3 min in 50 1 SDS-PAGE buffer and centrifuging at 1000 x G. The immunoprecipitates (2000 to 10,000 cpm) and th radioiodinated initial bodies and erythrocytes (200,000 cpm) wer electrophoresed on 7.5 to 17.5% (w v) gradient polyacrylamide gels. Th gels were fixed in glass-distilled water:methanol:acetic acid (6:4:1), wer dried, and were exposed to Kodak X-Omat AR. film (Eastman-Kodak Rochester, NY) at room temperature for 48 hr to identify th radioiodinated initial body and erythrocyte polypeptides, and at -70°C b using Cronex Quanta III intensifying screens (DuPont, Wilmington, DE for 72 hr to identify the immunoprecipitates. I4 C-Labeled proteins use for m.w. comparison (Amersham, Arlington Heights, IL) consisted of myosin, 200,000 m.w.; phosphorylase b, 92,500; bovine serum albumin, 69,000; ovalbumin, 46,000; carbonic anhydrase, 30,000; and lysozyme, 14,300. If the hybridoma supernatant contained monoclonal antibodies to Aml05, a band on the X-ray fil (autoradiograph) is observed at a molecular weight of about 105,000. Two cell lines (ANA 15D2 and ANA 22B1) were identified that produced anti-Aml05 antibodies. The cell lines were double cloned and the supernatants concentrated to 0.1 mg immunoglobulin/ml following determination of isotype (both were IgG3). The concentrated supernatants were used for all further testing. The immunoprecipitation of ^I-AmlOS was repeated with the double cloned hybridoma supernatants. The evidence that Aml05 is an initial body protein and not of erythrocyte origin includes unreactivity of ANA 15D2, ANA 22B1, or rabbit anti-Aml05 antibodies (all positive on initial bodies in infected erythrocytes) with uninfected erythrocytes or infected erythrocyte membranes using immunofluorescence, and failure of these antibodies to immunoprecipitate 12S I radiolabeled erythrocyte ghosts. Additionally, ANA 15D2 AND ANA 22B1 immunoprecipitate Aml05 metabolically radiolabeled in vitro during short term erythrocyte culture. It has been demonstrated that during short term cultures 35 S incorporation occurs exclusively in initial bodies. Aml05 is immunoprecipitated as a doublet i band seen most clearly with ^S labelled Aml05 or silver stained Aml05. 2 The doublet is consistently present and has been found to be indicative 3 of a complex of two Anaplasma marginale proteins having surface-exposed 4 epitopes. The two proteins as a complex are herein sometimes referred 5 to as Major Surface Protein 1 (MSP-1) as well as the term Aml05. 6 The two proteins which make up the doublet are herein referred to as 7 Aml05U and Aml05L. The protein Aml05U is also sometimes referred 8 to as MSP-la with the Aml05L sometimes referred to as MSP-lb. The 9 proteins forming the complex have electrophoretic mobilities which are 10 very nearly the same. Additional testing has indicated that Aml05U has u electrophoretic mobility corresponding to molecular weight of 12 approximately 105 kilodaltons. Some measurements have indicated the 13 electrophoretic mobility of Aml05L is more nearly 100 kilodaltons. 14 These mobilities are associated with the proteins of the MSP-1 or 15 Aml05 complex for the Florida geographical isolate of Anaplasma 16 marginale. As further described below the corresponding proteins for 17 other geographical isolates of Anaplasma marginale show a high degree 18 of polymorphism in the proteins which make of the MSP-1 complex. 19 Accordingly, the electrophoretic mobilities of the proteins forming this 20 complex vary. However, the antigenic nature of these different isolates 21 is similar as will be explained hereinafter. Monoclonal antibodies can in general be prepared against other antigenic surface proteins of Anaplasma marginale using procedures the same as or similar to those described above. To date hybridoma cells producing monoclonal antibodies have been created against additional antigenic surface proteins of Anaplasma marginale. TABLE 1 Antigen Monoclonal Antibodv and Cell Tvpe Aml05 (complex) ANA 15D2(15D2), ANA 22B1(22B1) Aml05 (complex) F34C1 Am86 AMG75C2 Am36 F19E1, ANAO-58A2 Testing for Neutralization of Anaplasma marginale by Monoclonal Antibodies- In order to test for initial body neutralization using the two anti-Aml05 monoclonal antibodies, 10 10 initial bodies were incubated with pooled ANA 15D2 and ANA 22B1 ascitic fluid, and the mixture was injected into splenectomized calves. Although complete neutralization was not observed, a significant prolongation of the prepatent period occurred (Table 2). The experiment was repeated with the assumption that the prolonged prepatent period observed with 10 10 initial bodies indicated partial neutralization and that complete neutralization would likely occur at a lower infective dose. In the second experiment complete neutralization of initial body infectivity occurred using 10 7 initial bodies and partial neutralization as judged by prolonged prepatent periods occurred using 10*, 10 9 , and 10 10 initial bodies. The mechanism of neutralization by the anti-Aml05 monoclonal antibodies is unknown at present. Certainly monoclonal antibodies directed against an initial body determinant necessary for erythrocyte receptor binding would neutralize infectivity. ANA 15D2 and ANA 22B1 may recognize the same or overlapping determinants as they reciprocally inhibit binding of each other to , 5 I-Aml05 in a competition radioimmunoassay. Other modes of neutralization include agglutination and when possible across murine-bovine species lines, interaction with bovine effector cells and complement-fixation with initial body lysis. The effective use of Aml05 as a protective immunogen to prevent bovine anaplasmosis wc- d require that the determinants recognized by the neutralizing monoclonal antibodies be common to all isolates in a given region. Both similarities and differences in protein and antigeni composition among various isolates of Anaplasma marginale have been demonstrated. Six isolates from widely geographically separated areas of the U.S. (Florida; Okanogan, Washington; South Idaho; North Texas; Clarkston, Washington; Virginia) have been examined for the presence of determinants recognized by ANA 15D2 and ANA 22B1 using indirect immunofluorescence on acetone fixed blood smears. The determinants were present on 100% of the initial bodies (as determined by comparison with Wright's stained initial bodies in an adjacent section of the smear) in all six isolates. Additionally, the determinants were present at all stages of a primary, acute infection from 1% parasitemia through peak parasitemia with hemolytic crisis. The presence of thee determinants now identified as protective antigens on multiple isolates and their presence at all stages of infection fulfill important criteria for use of Aml05 or its fragments as a vaccine. Aml05 and Aml05 polypeptides containing determinants recognized by the neutralizing monoclonal antibodies have been tested as effective immunoprophylaxis for bovine anaplasmosis. Partial neutralization of infectivity of 10 10 Anaplasma marcpnale initial bodies by monoclonal antibodies (ANA 15D2/22B1)- BALB/c X B10A (3r) mice were injected intraperitoneally (i.p.) with 1.0 ml Pristane and one week later with 2-3 X 10 6 double cloned hybridoma cells suspected of producing anti-Am!05 antibodies. Ten 2030 -35- days after injection with the hybridoma cells, ascitic fluid was withdrawn from the mice, centrifuged to pellet debris, and passed over a glass wool column. The indirect fluorescent antibody (IFA) titer is determined. A strong titer would be 1:16,000. 10 10 initial bodies are purified from Anaplasma marginale (Florida isolate) infected erythrocytes as described in Palmer, G.H. et al, J. Immunology, 1331010 (1984) and resuspended in 2.5 ml RPMI 1640 (2mM L-Glutamine, 25mM HEPES). The initial bodies are added to 2.5 ml of ascitic fluid. The initial body-ascitic fluid suspension is briefly vortexed, incubated for 45 min. at room temperature, and injected into the left semitendinosus muscle of each cal. Daily blood samples are collected for 75 days post-inoculation (DPI) to determine packed cell volume (PCV) and parasitemia (1000 erythrocytes counted). Probability values (p) are calculated using the pooled t-test p values of less than 0.05 are considered significant NS, not significant. ND, significance not determined. The results of a test using two cell lines designated ANA 15D2 and ANA 22B1 which produce monoclonal antibodies to Aml05 and a cell line TRYP 1E1 which produced monoclonal antibodies against Trypanosoma brucei are reported in Table 2. 30 • 36- TABLE 2 Parameter ANA 15D2/22B1 TRYP 1E1 Significance No. infected/ 6/6 4/4 challenged Mean DPI* to 1% 31 (29-37) 24 (23-25) p<0.01 parasitemia (range) Mean peak para- 51 (30-58) 71 (69-71) P≤.0.01 sitemia (%) (range) Mean low PCV* 15 (13-17) 11 (10-11) NS (range) No. dead/challenged 0/6 3/4 ND 'DPI means days post inoculation. "PCV means packed cell volume. _ These results indicate that by using 10 10 initial bodies partia 2 neutralization was observed as judged by a significant prolongation of th J prepatent period. 4 Neutralization of infectivity of graded numbers of initial bodies b 5 monoclonal antibodies (ANA 15D2/22B1)- 6 The experiment was repeated with the hypothesis that th 7 prolonged prepatent period observed with 10 10 initial bodies indicated 8 partial neutralization and that complete neutralization would likely occu 9 at a lower infective dose. 10 The protocol used was similar to that described in Part a. u A constant amount of ascitic fluid was inoculated with either 10 10 , 10 9 12 10*, or 10 7 purified Anaplasma marginale (Florida) isolate initial bodies 13 injected into calves, and infection monitored as previously described 14 The results are reported in Table 3. TABLE 3 No. of Initial ANA 15D2 Parameter Bodies 22B1 TRYP 1E1 Significance No. infected/ 10 10 3/4 3/3 ND challenged 10 9 4/4 3/3 ND 10* 4/4 3/3 ND 10 7 0/4 3/3 ND Mean DPI to 10 10 33 (32-33) 26 (25-28) ρ_ 0.01 1% parasitemia 10 9 35 (33-39) 28 (27-29) p_<0.01 (range) 10* 37 (36-38) 30 (26-32) p O.01 10 7 neg @ 75 34 (34-36) ND These results indicate that complete neutralization of infectivity occurred using 10 7 initial bodies and partial neutralization, as judged by prolonged prepatent periods, occurred using 10*, 10 9 and 10 10 initial bodies. Purification of Monoclonal Antibodies- The steps for purifying the monoclonal antibodies to couple to th immunoadsorbent column are: 1. BALB/c X BIO A (3r mice were injected intraperitoneall with 1.0 ml Pristane (Aldrich Chemical Co., Milwaukee, WI) and on week later with 2-3 X 10* double cloned hybridoma cells. Ten day later, ascitic fluid with withdrawn from the mice, centrifuged 1,675 X G to pellet insoluble debris and passed over a glass wo column. 2. A 50% (v v NH 4 S0 precipitation is conducted on the wo column effluent and following resuspensioπ in .032M Tris is dialyzed f 24 hrs. against .032M Tris. This dialyzed sample is chromatographe on a DE-52 cellulose column and eluted using a 0-0.2M Na continuous gradient in .032M Tris. The eluted fractions are titere using indirect immunofluorescence on acetone-fixed smears of Anaplasm marginale infected blood. The purity of the antibody is determine using sodium dodecyl sulfate-polyacrylamide gel electrophores (SDS-PAGE) with detection of protein using Coomassie Blue staining. Preparation of the Monoclonal Immunoafϊinity Chromatograp Column- The purified monoclonal antibody, ANA 15D2 (pure by Coomass Blue staining on SDS-PAGE) is dialized against 0.1M NaHC0 3 , 0.5 NaQ pH 8.3. The dialized protein is coupled to CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ.) at lOmg protein/ml settled Sepharose 4B overnight at 4 C C by rotation. The coupled immunoaffinity matrix (ANA 15D2-Seph 4B) is poured into an Econo Column 11_5 ml column Bio-Rad Lab-, Richmond, CA) and stored at 4 β C until use. Source of the Anaplasma marginale Initial Bodies- Anaplasma marginale has never been successfully grown in long-term in vitro culture. The source of Anaplasma marginale therefore is blood of infected, splenectomized calves. The calves are inoculated with 10 10 initial bodies intramuscularly and then checked daily for evidence of parasitemia using Wright's stained blood smears. When the percentage of infected erythrocytes reaches 40-95%, 4-7 liters of blood are collected into 4 units/ml heparin sulfate. The erythrocytes are washed 3X with phosphate buffered saline, pH 7.2 and then resuspended 1:1 in 31.2% dimethylsulfoxide in phosphate buffered saline. This suspension is frozen in liquid nitrogen and constitutes the Anaplasma marginale initial body source. Disruption and Treatment of Anaplasma marginale Initial Bodies- The Anaplasma marginale infected erythrocytes (10 12 will yield approx. 1.0 mg pure Aml05) are thawed from liquid nitrogen at 37°C and washed 5X in phosphate buffered saline using centrifugation at 27,000 X G. The sediment is resuspended in 35 ml phosphate buffere saline, disrupted by 2 min. of sonication at 50 watts (127 X 4 m titanium probe, Braun-sonic 1510, Braun Instruments, San Francisco, CA and then washed two times at 1650 X G for 15 min. The purifie initial bodies are disrupted in 5 ml 50mM Tris-HCl (pH 8.0, 1.0 Nonidet P40, 0.1% sodium dodecyl sulfate, 5mM EDTA 5mM iodoacetamide, ImM phenlmethylsulfonylfiuoride, 0.1 mM tosyl-L-lysy chloromethane) and applied to the ANA 15D-Sepharose 4B monoclona immunoaffinity column. Column Chromatography- The ANA 15D2-Sepharose 4B monoclonal immunoaffinity colum is washed with 100 X column volume with TEN buffer (20mM Tris-HCl 5mM EDTA, 0.1 M NaCl, 25mM NaN 3 , pH 7.6) 1% NP-40, 0.1% SD and then approximately 10 12 disrupted initial bodies are loaded onto th column at a rate of 25 ml/hr. The unbound proteins are washed ou using 100 X column volume TEN with 1.0% NP-40 and 0.1% SDS an then any remaining unbound proteins and the NP-40, SDS (nondialyzabl detergents) are removed by 100 X column volumes TEN withou detergent The specifically bound Aml05 is eluted using 50mM Tris p 8.0 with 0.5% deoxycholate and 2M KSCN. Recovery of Purified Surface Antigen- The eluted protein (Aml05) is dialyzed against phosphate buffered saline to remove the KSCN and deoxycholate. Aml05 is quantitated using a modified Lowry protein assay and frozen at -70°C until use. Preparation of Vaccine- Aml05 is thawed and suspended in Freund's incomplete adjuvant to produce a vaccine in which purified antigen, such as Aml05, is present in an amount of 10, 25 or 100 micrograms/milliliter. Immunization Studies- Groups comprised of 5 Holstein calves, weighing approximately 100kg, were immunized 4 times with 100 g of either ovalbumin emulsified in Freund's complete adjuvant (group 1), Aml05 emulsified in Freund's incomplete adjuvant (group 2), or Am36 emulsified in Freund's incomplete adjuvant (group 3). The immunizations were conducted on day 1, day 17, day 35 and day 59. Group 4, which consisted of 4 calves, was not immunized. The antibody response of the 4 groups to Aml05 was determined using a radioimmunoassay based on 12S I-Aml05. The calves were challenged on day 83 with 10* purified Florida isolate A. marginale initial bodies. The calves were monitored for infection by daily clinical examination, determination of hematocrit, and - examination of Wright's .tamed bleed ■ „ __ preseπce of parasitM _- The results are presented in Table 4. ____ m m ___ ___ m _ m __ umm __________■_____ ■ _______ ■ ____________. TABLE 4 Group 1 Group 2 Group 3 Group 4 Titer to Aml05 10 2 10 4 10 2 0 Ho. infected/ No. challenged 5/5 3/5 3/5 4/4 _ days to infection 33 39(p\<.01)* 38 29 x peak parasitemia 5.4 <.01(p\<.01) 1.5 4.2 x low PCV* 24.4 31(p\<.01) 28 23 'Significance: p values were calculated by the pooled t-test Probability values of less than 0.05 were considered significant *PCV = packed cell volume. ___ -ιι ___ βι _ ι _______________ ι ___ ιι ____^^ The significant elongation of the prepatent period (days until infection was detected), significant reduction in parasitemia, significant difference in PCV, and complete protection in 2 of 5 Aml05 vaccinates relative to calves immunized ovalbumin indicates that Aml05 is capable of inducing significant protection against challenge with Anaplasma marginale. General Discussion- The major Anaplasma marginale initial body surface proteins and protein complexes identified to date (Aml05 complex, Aml05U, Aml05L, Am86, Amόl, Am36, Am31, Aml5) each have a surface exposed epitope on the initial body. Evidence for the surface nature of Aml05, Am86, Amόl, Am36, Am31, and Aml5 proteins was obtained by radioiodination of the proteins on intact initial bodies using a membrane impermeant radiolabeling technique (lactoperoxidase) (Palmer, GH, McGuire TC: J. Immunol 133:1010-1015, 1984). Anaplasma marginale initial bodies were purified from parasitized erythrocytes by using ultrasonic disruption and differential centrifugation. The initial bodies were intact as determined by electron microscopy, were not agglutinated by anti-bovine erythrocyte sera and were infective. The initial body proteins surface radioiodinated using lactoperoxidase included Am220, Aml05 complex, Aml05U, Aml05L, Am86, Amόl, Am56, Am42, Am36, Am31 and Am25. Of these Aml05, Aml05U, Aml05L, Am86, Amόl, Am3ό and Am31 are precipitated b 12030 -46- neutralizing antibody. The latter group of proteins are major surface proteins and one or more of these proteins alone or in combination might be incorporated in a vaccine or diagnostic test In fact, the data presented in Table 4 shows that either purified Aml05 or Am36 will induce protective immunity against virulent Anaplasma marginale challenge in calves. That purified proteins will work as vaccines indicates that similar results might be achieved with synthetic peptides of 6 amino acids or more mimicking the antigenic structure of the biologically active epitopes, with antigens expressed in heterologous bacteria containing the genes coding for the biologically active epitopes of the surface proteins or with one or more antigens expressed in virus vectors containing the genes coding for biologically active epitopes of the surface proteins. The major Anaplasma marginale initial body surface proteins (Aml05, Aml05U, Aml05L, Am86, Amόl, Am36 and Am31) bear epitopes recognized by neutralizing antibody. Antiserum prepared by immunization of rabbits with purified Anaplasma marginale initial bodies completely neutralized the infectivity of 10 10 purified initial bodies for splenectomized cattle. Using the technique of immunoprecipitation these proteins were recognized by the neutralizing antibody, demonstrating their potential roles, either individually or in combination, in inducing neutralizing antibody and therefore their use as an improved vaccine for cattle. The recognition of these surface proteins was consistent regardless of the isolate (Florida, Washington-O, Virginia) of used t immunize the rabbits to prepare the antiserum. It has been shown that Aml05, Aml05U, Aml05L and Am3 each bear an epitope common among Anaplasma marginale isolates teste (Florida, Washington-O, Washiπgton-C, Virginia N. Texas, S. Idah Kansas, Oklahoma, Kapiti (Kenya), and Israel-round and Israel-tails) an to Anaplasma centrale (a less virulent species) that are capable o inducing neutralizing antibody. The purification of Am 105 or Am36 b monoclonal antibody immunoaffinity chromatography and the demonstratio of its ability to induce protection in cattle immunized with the protei clearly shows their importance, either alone or in combination with othe surface proteins, as an improved vaccine against anaplasmosis. The Aml05, Aml05U, Aml05L or Am36 epitopes are completel protease sensitive and do not bear any carbohydrate residues and such can be easily mimicked by short (minimum 6 amino acid synthetic peptides or by polypeptides expressed in a foreign bacteriu or virus containing the gene coding for the epitopes. The availabili of monoclonal antibody makes both the synthetic peptide and the gen cloning procedures alternative approaches to vaccine development as explained more fully below. The surface proteins Aml05, Aml05U, Aml05L, Am86, Amό Am36, Am31, Aml3 identified to date are specifically recognized serum taken from cattle over a period of 30 days to 255 days post-infection. This recognition is consistent regardless of the isolate used to infect the cattle (Florida, Virginia, N. Texas). This specific recognition is required for selection of Anaplasma marginale proteins to be used individually or in combination as the antigen in an improved serologic assay to diagnose anaplasmosis in cattle. These supporting data point to use of these proteins for diagnosing anaplasmosis. The isolation and incorporation of these proteins into a serologic assay for diagnosis is a straightforward technical procedure. The findings to date also indicate potential use of a synthetic peptide of 6 amino acids or more or a polypeptide expressed in a vector organism as immunologically equivalent agents for diagnostic purposes. Aml05 and Am36, isolated by monoclonal immunoaffinity chromatography and coated into wells of a microtiter plate at 5 to 100 ng/well, have been tested as the basis of an Enzyme Linked Immunosorbent Assay (ELISA) for serologic diagnosis of anaplasmosis. Each assay has been found capable of differentiating non-infected from Anaplasma marginale or Anaplasma centrale infected cattle at periods from 30-255 days post-infection and was accurate regardless of the isolate used to infect the -attle (Florida, N. Texas, Virginia, Washington-O, Washington-C, Idaho, Kenya, Israel-round, Israel-tailed). The present serologic assay is based on the isolated whole Aml05 or 2 Am36. These findings, however, imply the potential use of a 2 immunologically similar synthetic peptide of six amino acids or more o 3 a polypeptide expressed in a vector organism. 4 Proteins of 105,000 daltons (Aml05), 86,000 daltons (Am8ό), 61,00 5 daltons (Amόl), 31,000 daltons (Am31) and 15,000 daltons (Aml5), al 6 identified as surface proteins, are strongly antigenic as evidenced b 7 antibody in Anaplasma marginale-infcctcd cattle. In addition, dilution 8 of the post-infection sera have high titers to Am86 and Aml 9 throughout infection, indicating a preferential response. Use of Am8 10 alone or in combination with Aml05, Amόl, Am31, or Aml5 as a u antigen in a diagnostic test is implied from these findings. The presen 12 stage of this research also points to potential use of an immunologicall 13 similar synthetic peptide of six amino acids or more, or a polypeptid 14 expressed in a vector organism which has epitopes recognized b 15 post-infection sera as antigens in diagnostic tests for anaplasmosis. 2<J 17 PART II - SIZE POLYMORPHISMS IN DIFFERENT GEOGRAPHICA 18 ISOLATES OF Anaplasma marginale 19 Additional research has indicated that there are significant siz 20 polymorphisms in the two proteins forming the MSP-1 protein compl 21 between different geographical isolates of Anaplasma marginale. Tabl 22 5 below shows the range of sizes for the corresponding compone - » „_.,_ PCT/US90/01678 2030 -50- proteins Aml05U and Aml05L for the geographical isolates of Anaplasma marginale for Florida (F), southern Idaho (I), northern Texas (T), Virginia (V), and Clarkston, Washington (W). These molecular weight estimates were made by electrophoretic mobility analysis using polyacrylamide electrophoresis as explained by Oberle et al., Infection and Immunity- Vol. 56, No. 6 (1988) which is hereby incorporated hereinto by reference. - .. 2 3 TABLE 5 4 Apparent Molecular Mass (kDa) in Isolate* 5 Major Complex . 6 Component F I T V W 9 AmF105 105 98 97 100 100 10 u AmFlOO 100 95 89 70 86 12 13 Recognized 14 by 22B, 105 95 89 70 86 15 16 Recognized 17 by R911 100 98 97 100 100 18 19 'Isolates: F, Florida; I, Southern Idaho; T, Northern Texas; V, Virginia 20 W, Clarkston, Washington. 21 PART m - RECOMBINANT Aml05L In this part we demonstrate that Aml05 consists of a complex of two noncovalently Knked polypeptides of similar molecular weight To determine whether these two polypeptides, termed Aml05U and Aml05L, are products of separate genes, and to examine the structural and antigenic relationships between the polypeptides, we cloned and expressed genes coding for Aml05L epitopes in Escherichia coli. In this report, we identify Aml05U and Aml05L as separate gene products, each bearing surface-exposed epitopes. Cloning and expression of Aml05L will allow determination of its efficacy as a single, non-complexed immunogen. Preparation of Antisera- Mouse monoclonal antibodies were prepared as described before (14, 17) and designated as follows: 1E : and 24A 1? control antibodies to a surface glycoprotein of Trypanosoma brucei; F19E, an antibody that immunoprecipitates Am36 (19); 15D 2 and 22Bι, antibodies that immunoprecipitate Aml05 and neutralize infectivity of Anaplasma marginale in vitro (17); and F34C,, an antibody that immunoprecipitates Aml05. Antisera to Am 105 (17), to isolated Anaplasma marginale initial bodies (19), and to E. coli containing pBR322 or pAM25 plasmid DNA were made in rabbits. Rabbits were immunized four times with lysed bacteria (2 x 10 9 organisms in complete Freund adjuvant for the first immunization, and 10 10 organisms in incomplete adjuvant for the other three). Titers were evaluated by an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (3), or immunoprecipitation of [ M S]methionine-labeled extracts of Anaplasma marginale(2). These rabbit antisera are designated as follows: R612, a control antibody prepared against a surface glycoprotein of Tbrucei; R781, an antibody prepared against isolated initial bodies of Anaplasma marginale; R873 and R874, antibodies prepared against Aml05 isolated by immunoaffinity chromatography on monoclonal antibody-Sepharose 4B (17) purified Aml05 consists of Aml05U and Aml05L); R907, an antibody prepared against E. coli(pBR322); and R911, an antibody prepared against E. co/ι(pAM25). Antigen detection on nitrocellulose filters- Proteins of Anaplasma marginale or recombinant E. coli were bound to nitrocellulose filters and detected by reaction with specific antisera and 125 I-labeled protein A as described by Young and Davis (26), with two modifications: (i) after chloroform lysis, filters were fixed in 10% acetic acid-25% isopropaπol; and (ii) 1% hemoglobin was added to buffers instead of bovine serum .ilbumin to block nonspecific binding of 12S I-labeled protein A to the filters. ELISA- ELISAs were as described by Ellens and Gielkens (6), using Aml05 attached to plates at 50 ng per well. The enzyme label was horseradish peroxidase-protein A, and the substrate was recrystallized 5-aminosalicylic acid. Aml05 was isolated from Anaplasma marginale by immunoaffinity chromatography on monoclonal antibody 15D 2 -Seρharose 4B (17) and consisted of Aml05U and Aml05L. Sera against Aml05 and against E. coli containing pBR322 or pAM25 were prepared in rabbits. Immunoprecipitation- Anaplasma marginale organisms were radiolabeled by metabolic incorporation of [^Sjmethioπine during short-term in vitro culture (2) or by surface radioiodination, using lactoperoxidase (19). E. coli organisms were also labeled with S during exponential growth in 1-ml cultures containing 250 Ci of [^Sjmethionine and 35 g of ampicillin per ml. After removal of the unincorporated radiolabeL, organisms were solubilized by sonication at 4°C in a lysis buffer consisting of 50 mM Tris hydrochloride (pH 8.0), 5 mM EDTA, 5 mM iodoacetamide, 1 mM pheπylmethylsulfonyl fluoride, 0.1 mM N-tosyl-L-hsine chloromethyl ketone, 0.1% (wt vol) sodium dodecyl sulfate (SDS), and 1% (vol/vol) Nonidet P-40. The -solubilized extract was centrifuges at 130,000 x g for 1 h at 4 β C and passed through a 0.2- -pore-size filter (Centrex; Schleicher & Schuell, Inc.) before being used for immunoprecipitation with rabbit or mouse antibodies and protein A-bearing Staphylococcus aureus (Calbiochem)(9, 17, 23). The precipitated radiolabel was eluted and analyzed on 7.5 to 175% polyacrylamide-SDS gels, 7.5% polyacrylamide gels containing 4M urea, or 5% polyacrylamide gels containing 4 M. 1 C-labeled standard proteins were as follows (molecular weight): myosin (200,000), phosphorylase b (92,500), bovine serum albumin (69,000), ovalbumin (46,000), carbonic anhydrase (30,000), and lysozyme (14,300). For the experiment described below (see Fig. 5) immunoprecipitated recombinant Aml05, Aml05U, and Aml05L protein bands were cut out from dried 7-5% polyacrylamide-4 M urea gels and then separately rehydrated and electroeluted into a mixture of 50 mM Tris hydrochloride pH 8.0) 0.1% (wt vol) SDS, and 1% (vol/vol) Nonidet P-40. Polyacrylamide was removed by centrifugation, and the 35 S-labeled proteins were immunoprecipitated again from electroelution buffer. Peptide Mapping- Immunoprecipitated, 35 S-labeled proteins were cut out from dried polyacrylamide gels and compared for sequence homology by peptide mapping as described before (5). Radiolabeled peptides produced by limited proteolysis with S. aureus V8 protease were separated on 15% polyacrylamide-SDS gels and detected by fiuorography (4). Isolation of Anaplasma marginale DNA- Bovine blood, infected with Anaplasma marginale at >50% erythrocytic parasitemia, was washed four times with phosphate-buffered 2 saline. At each wash an upper layer containing leukocytes and 2 erythrocytes was removed. The remaining erythrocytes were then frozen 3 in phosphate-buffered saline at a packed cell volume of 50%. A 4 100-ml volume of the erythrocyte suspension was thawed and centrifuged 5 at 30,000 x g for 20 min at 4°C to pellet Anaplasma marginale initial 6 bodies and erythrocyte membranes. The pellet was washed a further 7 three times in phosphate-buffered saline at 30,000 x g to remove * hemoglobin from the lysed erythrocytes. DNA was then extracted from 9 initial bodies (11) and further purified by deproteinization with phenol- 10 chloroform, digestion with RNase A and proteinase K, and precipitation il with ethanol. 12 Preparation of Recombinant DNA Libraries- 13 Anaplasma marginale DNA was partially digested with Sau3A to 14 an average size of 5 kilobases (kb). Digested DNA was ligated with 15 BamHi-cleaved and dephosphorylated pBR322, using T4 DNA ligase (25). 16 E. coli HB101 cells were transformed to ampicillin resistance by the 17 high-efficiency transformation protocol and Hanahan (8). Plasmids 18 pAM22 and pAM25 were identified by expression screening of a library 19 containing 8,000 recombinants with R873 serum (rabbit anti-[Aml05U plus 20 Aml05L] complex). Other colonies in this library, sv ~ h as that 21 containing pAM14, also reacted with R873 and contained the pAM22 sequence plus various lengths of additional DNA that extended beyon the Bglll sites. A second library of 3,000 recombinants was prepared by digestin Anaplasma marginale DNA to completion with Bglll and ligating into th BamHI site of pBR322. Clones containing pAM97 and pAM113 wer identified in this library by expression screening with R873. Southern blotting- The protocol used was a modification of tha described by Wahl et al. (24). Portions (0.5 g) of Anaplasm marginale genomic DNA or plasmid DNA (0.36 g) were digested wit the appropriate restriction enzymes. For comparison of plasmid an genomic sequences on the same gel, 0.5 g of digested genomic DN or 1.8 ng of plasmid DNA was subjected to agarose gel electrophoresi and blotted onto nitrocellulose filters. Hybridization was at 65°C i 5x SSPE (0.18 M ' NaCl, 0.01 M NaH 2 P0 4 , 0.001 M EDT [pH 7.4])-0.25% Sarkosyl (Sigma) containing 10% dextran sulfate, 100 of denatured calf thymus DNA per ml, and a 32 P-labeled nick-translate probe. Filters were washed a total of five times, finally in O.l SSPE-0.0033% Sarkosyl at 65 β C. The probe was the 1.4-kb Ss fragment of pAM97, isolated from agarose gels. Genomic libraries and AMI 05 expression by __.. coli - Initi experiments investigated the specificity and sensitivity of immunobl assays in detecting Anaplasma marginale proteins immobilized o nitrocellulose filters (26). In previous studies we prepared monoclonal and polyvalent antisera against Anaplasma marginale which has specificity for different surface proteins (17-19). The reactions of these antisera with positive and negative control antigens are shown in Fig. IA. All antibodies detected Anaplasma marginale erythrocytes and did not react with noninfected erythrocytes. The sensitivity of detection was greatest with R873, a rabbit antiserum against immunoaffinity-isolated Aml05. R873 detected as few as 1,200 parasitized erythrocytes in the 1 microliter spot applied to the filter. The specificity of each antibody in immunoblots was the same as that observed previously in immunoprecipitation experiments. Polyvalent or monoclonal antibodies against Aml05 or another surface protein, Am36, reacted with the appropriate protein; there were not cross-reactions or reactions with the negative control, ovalbumin. R873 detected a minimum of 1 ng of purified AM105. R781 was an antiserum prepared against isolated Anaplasma marginale initial bodies; it immunoprecipitated both Aml05 (Aml05U and Aml05L) and Am36 (data not shown), and recognized Aml05 and Am36 in immunoblots (Fig. IA). We considered this assay sufficiently sensitive and specific to detect expression of Anaplasma marginale proteins in recombinant E. coli. Previous data have suggested that gene regulatory sequences of rickettsiae may function in E. coli (10, 13, 25). Accordingly, parasite 2 DNA was extracted from bovine erythrocytes containing a Florida isolate 2 of Anaplasma marginale. The DNA was partially digested with Sau3A, 3 inserted into the BamHI site of phosphatase-treated pBR322, and used 4 to transform E. coli HB101 to ampicilliπ resistance. This genomic 5 library was screened with R873 in the immunoblot assay for expression 6 of Aml05 antigenic determinants. 7 E. coli colonies containing recombinant plasmids of various sizes * reacted stably with the antiserum (Fig. IB). The restriction enzyme 9 maps of insert DNAs from pAM22 and pAM25, the smallest plasmids 10 of expressing colonies (3.75 and 4.15 kb, respectively), are shown in u Fig. 2. All plasmids from expressing bacteria contained the inserted 12 sequence present in pAM22; there were various lengths of additional 13 insert DNA in the larger plasmids which expended beyond the Bglll 14 sites. Restriction enzyme mapping and Southern blotting suggested that 15 the shaded sequence of 240 base pairs in pAM25 was not contiguous 16 with the remainder of pAM25 DNA in the Anaplasma marginale genome 17 and that two Sau3A fragments were ligated in this plasmid during 18 cloning. Both possible insert orientations with respect pBR322 DNA 19 were found in plasmids from expressing colonies (Fig. 2). 20 Analysis of each expressed plasmid DNA, and of genomic DNA 21 by Southern blotting, suggested that the inserted sequence in pAM22 22 should be contained within a single Bglll fragment of Anaplasma marginale genomic DNA. To confirm this, we prepared a second library. Anaplasma marginale DNA was digested to completion with Bglll and inserted into the BamHI site of pBR322. Plasmids pAM97 and pAM113 were identified in this library by expression screening with R873; they contained the expected Bglll fragment in both orientations (Fig. 2). Proteins Expressed by Recombinant E. coli - To characterize novel proteins synthesized by recombinant E. coli, bacteria containing either pAM25 or pBR322 were radiolabeled by metabolic incorporation of [^Sjmethionine. The radiolabeled proteins were analyzed by immunoprecipitation and SDS gel electrophoresis. The protein profile of recombinant E. coli is shown in Fig. 3, lane 7, and may be compared with the analogous profile of control bacteria containing only pBR322 (lane 8). All protein bands were present in both lanes, except for a major radiolabeled polypeptide of 105,000 molecular weight in recombinant bacteria. When labeled proteins were immunoprecipitated by R873, one normal E coli protein was recognized. However, in " recombinant bacteria, the additional 105,000-molecular-weight protein was also precipitated (compare lanes 5 and 10). A similar result was obtained with a different antiserum to Aml05, R874 lanes 3 and 12). These results demonstrated that a novel protein, coded for by pAM25 DNA, was expressed as a major component of the recombinant bacteria. This protein had a similar molecular weight and shared antigenic determinants with immuπoaffiπity-isolated Aml05 from Anaplasma marginale. R873 and R874 reacted with one or two normal E. coli proteins when used undiluted in immunoprecipitation, presumably because of prior exposure of rabbits to the bacterium. The possibility of a cross-reaction between AM105 and E. coli proteins is considered less likely, becaus antisera to lysed no recombinant E. coli did not recognize Aml05 (see Fig. 5 and 6). The reaction of R873 with E. coli was not observe in immunoblot ' assays because the dilution of antiserum used 1:4,000 effectively removed anti-E. coli activity while retaining activity agains Aml05. The molecular weight of the recombinant protein was identical i bacteria containing pAM25, pAM22, pAM97, or pAM113 plasmids. Th level of expression in each of these recombinants was also comparable as judged by relative band intensity on SDS gels. The orientation o insert DNA with respect to pBR322 had no apparent effect o expression (both orientations were equally represented in the fou plasmids. These data suggest the following: (i) that the Anaplasm marginale gene is functional in £. coli; (ii) that the gene is containe within the cloned Bglll fragment; and (iii) that the expressed molecul is not a fusion protein composed of both pBR322- and Anaplasma marginale-encoded amino acids. Recombinant Aml05 is structurally homologous to nonrecombinant Aml05L. Recombinant Aml05 was recognized by R873 and hence was antigenically homologous with Aml05U and/or Aml05L polypeptides. However, recombinant Aml05, expressed by any of the recombinants, was not recognized by monoclonal antibodies 22Bj or 15D 2 in immunoprecipitation or immunoblot assays (data not shown), or by R781 (Fig. 3, lanes 2 and 13). There were, therefore, important antigenic differences between recombinant and native Aml05. We compared recombinant Aml05 for structural homology with each component of the Aml05 doublet, Aml05L and Aml05U. Anaplasma marginale was radiolabeled with [ 35 S]methionine, solubϋized, and immunoprecipitated with the neutralizing monoclonal antibody 22B„ and the precipitated proteins were separated by electrophoresis in a 7.5% polyacrylamide-SDS gel containing 4 M urea (Fig. 4A, lane 3). The Aml05 doublet was clearly resolved. No bands were visible in the control lane {Anaplasma marginale plus 24A, monoclonal antibody, lane 4). Recombinant Aml05, immunoprecipitated by R873, was analyzed on the same gel. The recombinant Aml05 migrated as a single band in an identrcal position to Aml05L (Fig. 4A, lane 1). The Aml05 doublet in this gel system was resolved sufficiently to allow cutting out of the Aml05L and Aml05U components of the immunoprecipitate from a dried gel. Gel fragments containing each polypeptide were then rehydrated and analyzed by peptide mapping (5). Recombinant Aml05, immunoprecipitated by R873, was also cut out and analyzed. Figure 4B shows a peptide map obtained by partial digestion of the eluted polypeptides with S. aureus V8 protease. Cleavage peptides of recombinant Aml05 closely resembled those of Aml05L. Initial proteolysis products of both recombinant Aml05 and AM105L were polypeptides of 75,000, 59,000, and 51,500 molecular weight. Identical low-molecular-weight components (34,300, 18,600, and 13,000 to 16,000) were also generated. Therefore, the recombinant Aml05 and AM105L molecules were homologous and possibly identical. In contrast cleavage peptides produced from Aml05U were largely dissimilar to both Aml05L and recombinant Am 105. Predominant digestion products of Aml05U in the 22,000- to 27,000-molecular-weight range had no counterpart in Aml05L or recombinant Aml05. Another peptide of 16,000 molecular weight was also absent from Aml05L and recombinant Aml05. Although different peptides were generated from Aml05L and AM105U by proteolysis, the sensitivity of this procedure did not permit a determination of total nonhomology between Aml05L 2 and Aml05U. For example, cleavage peptides of 29,500 were produced 2 from both Aml05L and Aml05U. Whether these two 3 low-molecular-weight peptides share homology will require further 4 structural analysis. 5 Antigenic relationships among recombinant Aml05, Aml05L, and 6 Aml05U polypeptides. The antigenic relationships among Aml05L, 7 Aml05U, and recombinant Aml05 were investigated by preparing antisera 8 against bacteria expressing recombinant Aml05 in four rabbits; another 9 four rabbits were immunized with E. coli containing pBR322 as a 10 control. Sera were tested for recognition of noπrecombinant Aml05 by u an ELISA. All rabbits immunized with recombinant bacteria developed 12 antibodies to Aml05, ranging in titer from 1:100 to 1:1,000. No rabbits 13 immunized with control E coli developed antibodies to Aml05. The 14 anti-recombinant-Aml05 sera immunoprecipitated both Aml05L and 15 Aml05U from [ 35 S]methionine-labeled Anaplasma marginale (data not 16 shown), and therefore reacted similarly to R873 and 22Bj antibodies. 17 There are two possible explanations for these results. First, 18 Aml05L and Aml05U may share antigenic determinants and therefore 19 be immunoprecipitated together. Second, Aml05L and Aml05U may be ■ 20 antigeπically unrelat-.d but complexed. To discriminate between these 21 possibilities, Aml05L and Aml05U were separately purified and 22 immunoprecipitated. A detergent extract of [ 3S S]methionine-labeled Anaplasma marginale was first immunoprecipitated with monoclonal antibody 22B„ and the Aml05L and Aml05U components of the precipitate were separated by SDS gel electrophoresis. The Aml05L and Aml05U bands were cut out, electroeluted, and then separately immunoprecipitated again with monoclonal antibody 22B ! or with rabbit anti-recombinant-Aml05 serum (Fig. 5). Only Aml05U was reimmunoprecipitated by 22B,; Aml05L was not recognized (lanes 4 and 5). In contrast, anti-recombinant-Aml05 serum immunoprecipitated Aml05L but not Aml05U (lanes 8 and 9) when the two components were separated before immunoprecipitation. Therefore, recombinant Aml05 was antigenically homologous only to Aml05L. Thus, Aml05 exists as a complex of two polypeptides, Aml05L and Aml05U. Monoclonal antibody 22B, recognizes an epitope present on Aml05U, and binding to that epitope causes precipitation of both components of the complex. The complex is stable in 1% Noπidet P-40 and 0.1% SDS, which are present in the immunoprecipitation reaction, but is dissociated by boiling in SDS gel sample buffer. Aml05L and Aml05U are apparently not linked by disulfide bonds, because the molecular weight is unchanged when electrophoresis is performed under reducing or πonreduciπg conditions. Recombinant Aml05 is structurally and antigenically homologous to Aml05L. No evidence was obtained for structural or antigenic homology between 2 recombinant Aml05 and Aml05U polypeptides or between Aml05L and 2 Aml05U. These data explain the positive reaction of recombinant 3 Aml05 with rabbit anti-Aml05 sera and a negative reaction with 4 monoclonal antibody 22B-. 5 Surface radiolabeling of Anaplasma marginale initial bodies labels 6 both Aml05L and Aml05U. Viable initial bodies were radiolabeled with 7 12S I, using lactoperoxidase as described before (19). Labeled extracts * were then immunoprecipitated with R911 (anti-recombinant Aml05), R873 9 (anti-Aml05), monoclonal antibody 22Bι, or the appropriate control lo antibody. The precipitates were analyzed on polyacrylamide gels u containing 4 M urea (Fig. 6). The results showed that both Aml05L 12 and Aml05U polypeptides contained the radiolabel and were precipitated 13 by R911, R873, and 22B j . The increased band intensity of Aml05U 14 when precipitated by 22B, and of Aml05L when precipitated by R911 15 suggests some dissociation of the Aml05L-AM105U complex during this 16 immunoprecipitation. 17 Anaplasma marginale genome contains multiple copies of the cloned 18 Bglll fragment Anaplasma marginale genomic DNA was cut with 19 restriction enzymes; the DNA fragments were separated by gel 20 electrophoresis, blotted to nitrocellulose, and probed with 32 P-labeled 21 plasmid insert DNA from bacteria expressing recombinant Aml05. By 22 using enzymes which did not cut within the probe sequence, we observed multiple hybridizing bands (Fig. 7A, lanes 7 and 8). T discover whether these represented partially homologous copies of th cloned sequence or polymorphism in flanking regions, we cleaved genomi DNA with restriction enzymes that would generate a predictabl fragment HincII plus Mlul digestion should yield a 2.8-kb fragmen hybridizing to the HincII-Hindlll probe. For comparison, plasmid DN containing the entire 3.9-kb Bglll fragment was also digested with HincI plus Mlul and analyzed in the adjacent gel lane (Fig. 7A, lanes 5 an 6). The expected 2.8-kb fragment was found in both digests, bu hybridizing bands of 4.0 and 67.7 kb were also observed in the genomi DNA. The 4.0- and 6.7-kb bands must represent partially homologou copies of the 3.9-kb cloned Bglll fragment that do not have the HincI or Mlul site or both. Similar digests with HincII plus Bglll or SstI Bgll, or Bglll alone always produced the DNA fragment expected fro the map in Fig. 2, but with between two and four additional hybridizin bands (Fig.7A and B). Multiple hybridizing bands were detecte whether the HincII-Hindlll or SstI probes were used in detectio (Fig. 7B, lanes 2 and 5). There was no hybridization between clone probe and bovine leukocyte DNAs (Fig. 7B, lane 4), furthe demonstrating the parasite origin of the cloned sequence. Thus, the cloned DNA faithfully represents an Anaplasma marginale genomic sequence. However, additional partially homologous copies of the clones 3.9-kb Bglll fragment are also present in the genome. The data presented describe the expression of an Anaplasma marginale protein of approximately 105,000 molecular weight in recombinant E. coli. The antisera prepared in rabbits against immunoaffinity-isolated, nonrecombinant Aml05 recognize recombinant Aml05 and vice versa, showing shared epitopes. Also, antisera against recombinant Aml05 react with Anaplasma marginale in immunofluorescence and agglutinate purified initial bodies, demonstrating the presence of recombinant Aml05 epitopes on the parasites themselves. Recombinant Aml05 is structurally and antigenically homologous to Aml05L; no evidence was obtained for homology to Aml05U. Nonrecombinant Aml05, containing both Aml05L and Aml05U, confers protection on cattle against challenge with Anaplasma marginale (17). It is not known whether Aml05L or Aml05U, used separated as an immunogen, would confer protection. Aml05L and Aml05U are both accessible on viable initial bodies to surface radiolabeling, one important criterion for an immunoprotective protein (1). Aml05U may oe more likely to induce protection bec_.use this polypeptide contains the epitope recognized by neutralizing monoclonal antibody 22Bι (Fig. 5). However, other neutralization-sensitive epitopes may also be present in Aml05L, The epitope recognized in Aml05U by antibody 22B, is conserved in eight geographically distinct isolates (17), an important practical concern for potential immunization. Rabbit anti-recombinant-Aml05 sera also reacted with all isolates tested in immunofluorescence, but variation in surface-exposed epitopes might not be revealed by such polyvalent sera. Examination of the Anaplasma marginale genome by Southern blotting suggests the presence of a family of Aml05L genes and the possibility of antigen variation. A single Aml05L gene copy was detected in recombinant libraries by expression screening. Other copies of the gene may not be complete and functional, similar to pilin genes of Neisseria gonorrhoeae (15, 16, 22). Alternatively, other Aml05L genes may (i) contain promoter sequences that do not function in E. coli or Anaplasma marginale or (ii) code for antigenically variant forms of the protein not detected in the expression assay. An Aml05L-related gene could code for Aml05U, as peptide maps do not exclude the possibility of limited homology between Aml05L and Aml05U. However, later testing has indicated that the proteins Aml05L and Aml05U are the products of separate Anaplasma marginale genes as explained more fully below. Experiments in progress examine whether recombinant Aml05 will induce protection in cattle against disease and whether Aml05U may be expressed in E. coli so that both components of the Aml05 complex may be tested for protection. Immunoblot experiments and that shown in Fig. 5 demonstrate that the epitope on Aml05U recognized by neutralizing monoclonal antibody 22Bj is not denatured by solvents such as 2% SDS, 2-5% mercaptoethaπol, 10% acetic acid, and 25% isopropanol. Hence, this epitope is relatively resistant to conformational changes compared with, for example, surface-exposed epitopes of Trypanosoma brucei (4a). Other data suggest that immunoaffinity-isolated Aml05 is not glycosylated and show that the epitope recognized by antibody 22B- is protease sensitive (G.H. Palmer, S.D Waghela, W.C. Davis, A.F. Barbet, and T.C McGuire, Int J. Parasitol., in press). Expression of the Aml05U neutralization-sensitive epitope should, therefore, be readily obtained by direct monoclonal antibody screening of a fusion protein expression library, e.g., in bacteriophage lambda- gtll (26). In those libraries, . expression of Aml05U epitopes would not depend on recognition of rickettsial regulatory DNA sequences by E. coli (21). The most effective vaccine against Anaplasma marginale may be a combination of surface proteins. These include Am86, Amόl, Am36, and Am31 as well as Aml05, Aml05L or Aml05U. We described here the cloning and expression of an Anaplasma marginale gene in structural and antigenic homology between the cloned and native surface proteins. Since cattle are protected against Anaplasma marginale by immunization with Aml05 purified from infected erythrocytes (19), these results suggest that a recombinant vaccine is feasible and provide a rational basis for its development. PART IV - CLONING OF MSP-1 GENES FOR DIFFERENT GEOGRAPHICAL ISOLATES, DNA AND AMINO ACID SEQUENCES THEREFOR To characterize the MSP-la gene associated with the expression of Aml05U from widely different isolates we chose the approaches and procedures for cloning and sequence analysis which are described below. Such procedures should also be interpreted in light of other procedures referenced or described herein. For analysis and sequencing the foDowing geographical isolates of Anaplasma marginale were selected: Florida (FL) and Virginia (VA) isolates because they express the largest and the smallest polypeptide of the available isolates, respectively, and because our prior immunologic and molecular data on MSP-1 were obtained with FL as generally described hereinabove. Idaho (ID) isolate was chosen because it appeared the most variable by restriction analysis. Washington (WA) isolate because it was used in successful cross-challenge experiments of animals immunized with FL MSP-1 complexes. For sake of convenience in discriminating the particular geographical isolates being referred to, the antigens may hereinafter be referred to by the "Am" designation as used above, with an additional abbreviation such as "F* for Florida, to designate the geographical isolate. For example, the Aml05 generally referred to above in this application is also referred to as AmF105 to indicate the association with the Florida isolate. To begin analysis we first created a pseudo-random genomic library from Anaplasma marginale Florida isolate DNA by partial digestion with the restriction enzyme Sau3A. The resulting genomic DNA fragments were modified by adding additional C-tails thereto. The resulting modified DNA fragments were inserted into plasmids pUC9 which had been previously cleaved using the restriction enzyme PstI and G-tailed. The resulting recombinant plasmids which were then used to transform E coli (strain TB1). The resulting transfoπnant bacteria were screened with m I protein A, and monoclonal antibody 22B1 for expression of AmF105 epitopes. A portion of the MSP-la gene for the Florida isolate which codes for a subunit of AmF105U was obtained in a 2.7 kilobase pair (kbp) insert cloned into the plasmid pUC9 to produce a plasmid herein termed pAMTl. When plasmid pAMTl was inserted into E coli it caused the synthesis of an antigen containing a subunit of AmF105U having an approximately 56,000 dalton molecular weight. The portion of the AmF105U antigen expressed by this recombinant bacterial cell is indicated in the amino acid sequence information given in Fig. 12 for the Florida isolate starting with amino acid 1 through approximately 220-230 in repeat 8. To determine the number of MSP-la gene copies in the chromosome of the Florida isolate of Anaplasma marginale, Southern blot analyses of restriction endonuclease-digested Anaplasma marginale genomic DNAs were performed using the 2.7 kbp insert of pAMTl as a DNA hybridization probe. In most instances, only a single band hybridized with the probe, suggesting a single gene copy. Thus, the size polymorphisms which exist between the different Anaplasma marginale geographical isolates with respect to the corresponding MSP-1 protein complexes produced by each are probably due to allelic variations at one locus of the chromosome rather than, for example, by expression of different gene copies. Three other isolates of Anaplasma marginale were compared with FL by Southern blot restriction mapping, using the same probe. The restriction maps of all four isolates were virtually identical as shown in Fig. 8, with the exception of a variable length region between the internal Kpπl and Hindlll restriction enzyme cutting sites. This similarity in the restriction enzyme maps, as most easily seen with respeu to the 3' regions relative to the hatched regions indicating the gene, which confirms the position of each MSP-la allele at the same chromosome locus in each isolate. 2 The region of the plasmid pAMTl insert containing the partial 2 MSP-la gene was analyzed to determine its location which was found 3 to be in the region "right" of the Kpnl site (as seen in Fig. 8). This 4 analysis was performed by making progressive deletions in the "left" half 5 of the DNA insert of plasmid pAMTl using restriction enzymes and 6 monitoring the effects of those deletions on the in vitro synthesis and 7 size of the 56 kDa product encoded by pAMTl. * To isolate the intact MSP-ia gene from the FL isolate, we 9 created a random-sheared genomic library of Anaplasma marginale DNA lo using soπication of the isolated DNA. The sonicated DNA fragments u were blunt-ended using Kleπow fragment of DNA polymerase I. Ncol 12 linkers were thereafter added to the modified DNA fragments. The 13 resulting DNA fragments were ligated into the expression vector plasmid 14 pKK233-2. The resulting recombinant plasmids were implanted into 15 E. coli and the resulting bacterial cultures screened with monoclonal 16 antibody 22B1 for expression of the antigen AmF105 or antigens bearing 17 immunologically similar epitopes. The plasmid pKAna420 was identified 18 in the screening and further analysis of the expressed product by 19 electrophoresis and immunoprecipitation indicated that a fully-sized 20 iπ__nunoreactive prod .ct was being expressed. 21 For performing the DNA sequencing, we subcioned the Anaplasma 22 marginale DNA insert contained in pKAna420 into the Smal site of plasmid pGEM4 to create plasmid pFLlO and transformed the E. coli strain DH5a using pFLlO. Size-selected genomic libraries were then constructed from the DNA of the Virginia, Washington and Idaho Anaplasma marginale isolates by ligation of DNA fragments cut by the restriction enzyme Kpnl (for the VA isolate) or restriction enzymes Kpπl and PstI ( for the WA and ID isolates). The DNA fragments were then cloned into plasmid pGEM4 which had been linearized using enzymes Kpnl or Kpnl and PstI, and used to transform E. coli strain DH5a. The bacterial transformants were screened by colony hybridization according to the procedures of Grunstein and Hogness, Proceedings of National Academy of Sciences. CU.S.A.) 72. 3961 f!975\ The procedure was accomplished using a 1 Kbp DNA fragment of plasmid pAMTl radiolabeled with ^ as a hybridization probe, which was extracted from the Kpnl site (see corresponding point on Florida isolate restriction map) to the right end of plasmid pAMTl as shown in Fig. 8. The blocked-in or bolded linear regions of the plasmid diagrams shown in Fig. 8 correspond to the regions of the four geographical isolates of Anαplαsmα marginale which were DNA sequenced (see Fig. 10 for DNA sequences). The abbreviations used in Fig. 8 are as follows: Ac=AccI; A2=Av. Jϊ; B=BamHI; BclI=BclI (multiple restriction sites are shown for this enzyme for WA and FL isolates); Sm ~~ SmaI; Ss=SstI; and X=XmaIII. The fidelity of all four cloned fragments with the associated isolate Anaplasma marginale chromosome was confirmed by Southern blot comparisons of restriction fragments with genomic DNA (not shown) and by the expression of full-sized immunoreactive products by each transformant as shown by the Western blots of Fig. 9. The electrophoretic mobility tests indicated by Fig. 9 were produced from the recombinant E coli which were grown to an ^ ^ of 0-5-0.6 in L-broth containing 50 micrograms/milliliter ampicillin. They were collected by centrifugation and disrupted by boiling for 3 minutes in SDS-PAGE sample buffer, such as described by Laemmli, Nature (London), 227, 680 (1970). The polypeptides were fractionated on 7.5-17.5% gradient polyacrylamide gels, and transferred electrophoretically to nitrocellulose, and probed with monoclonal antibody 22B1 and 125 I-protein A. The bands containing the products of the MSP-la gene recognized by monoclonal antibody 22B1 are indicated by arrowheads. Molecular weight standards shown at the right of that Fig. are given in kilodaltons. Lanes 1,3,5, and 7 are recombinants pVAl, pWAOl, pID6 and pFLlO, respectively. Lanes 2, 4, 6, and 8 are polypeptides produced by VA, WA, ID and FL Anaplasma marginale initial bodies. DNA sequences for the four different geographical isolates o Anaplasma marginale were obtained as shown in Fig. 10. The DN inserts in the recombinant plasmids pGEM4 were sequenced using the dideoxynucleotide method of Sanger et al., Proceedings of National Academy of Sciences (U.S.A.) 82, 648 (1985). The SP6 and t promoter primers of pGEM4 were used to prime the initial sequencin reactions. Once into each insert, new primers were synthesized based on the sequences just obtained and used to extend the regio sequenced. The sequences are given from the 5' Kpnl site of eac clone to the same point representing the 3' end of the Florida isolat cloned insert Annotations above the sequences indicate the Kpnl site features of the promoter region, the start of transcription, the start an stop codons of the coding sequence, and the repeat units. Varian bases are indicated by asterisks beneath the sequence, whereas insertio or deletions are indicated by dashes. A region of homology near th 3 * end which is contained in repeat regions is double underlined ther and in the repeat regions. Further discussion of notable points abou the DNA sequences will be given below. The above descriptions of suitable methods for gene identificatio isolation, cloning and expression are also applicable to remaining antig- i according to this invention. More specifically, these techniques, wit suitable modification for the particular antigen being sought, may als 2 be used to create recombinant plasmids or other recombinant vectors 2 containing recombinant nucleic acids, DNA or RNA, coding for the 3 expression of purified antigenic proteins similar to or the same as the 4 native antigens indicated above for Anaplasma marginale. More 5 particularly, indicated antigens, such as Am220, Aml05 (complex), 6 Aml05U, Aml05L, Am86, Amόl, Am56, Am42, Am36, Am31 and Am25; 7 even more preferably Aml05 (complex), Aml05U, Aml05L, Am86, Amόl, * Am36, Am31, and Aml5; from the Florida isolate as used in the 9 research indicated above, or the antigenically similar proteins and 10 polypeptides from other isolates of Anaplasma marginale might be u produced by such recombinant techniques. Similarly, such recombinant 12 techniques may be applied to determine the DNA and/or polypeptide 13 sequences of the desired antigenic, and in applicable uses immunogenic, 14 proteins or polypeptides. The amino acid sequence information can then 15 be used to produce the antigenic polypeptides according to well-known 16 polypeptide synthesis techniques which are commercially available given 17 knowledge of the desired polypeptide sequence to be constructed. tl 18 should also be appreciated that the antigens, vaccines, recombinant 19 vectors and recombinant cells, methods and other aspects of this 20 invention are in there broader concepts applicable to the broader classes 21 of Rickettsiae since at least one member thereof is immunologically 22 treatable and detectible using the antigens and vaccines of this invention. This in particular applies to the more specific nucleic acid and amino acid sequences, described above and in more detail below, which are known effective for inducing an immune response against such parasitic organisms. Description of the MSP-la Gene Structure- Portions of each of the four DNA inserts of the recombinant plasmids pFLlO, pIDό, pWAOl, pVAl, and plasmid pAMTl were sequenced with the sequenced portions of the four isolate derived plasmids indicated by the bold lining in Fig. 8 and by the DNA sequences shown in Fig. 10, except pAMTl which is not shown in Fig. 10 because it is redundant with portions shown for pFLlO. To define the gene we first located the transcription start site. To do this, total cellular RNA from the Florida isolate Anaplasma marginale initial bodies was sequenced using a primer specific to a region near the 5' end of the only significant open reading frame (ORF), according to a procedure indicated by Vander Ploeg et al., Nucleic Acids Research 10, 3591 (1982). The RNA was sequenced directly with Avian Myeloblastosis Virus reverse transcriptase by a modification explained in Hollingshead et al, Molecular Cell Genetiics 207, 196 (1987), of the method of Inoue and Cech, National Academy of Sciences (U.S.A.) 82, 648 (1985). Synthesis of a runoff transcript ending at base 1FL (for base number 1 of the FL isolate sequence) identified this as the start of transcription (see Fig. 11A). The primer was the reverse complement of bases 147FL to 166FL (see Fig. 10). The presumptive promoter for the MSP-la gene was identified by its location relative to the transcription start site and by its striking homology with E. coli promoter consensus sequences. The promoter region structures of the different geographical isolate alleles and E. coli vary slightly as shown in Fig. 11B. The -35 and -10 region, and the start of transcription are indicated by a double underline in that Fig. Homologous bases between the E. coli consensus promoter and the Anaplasma marginale promoter sequences are indicated by holding. Lower case letters indicate bases not shared between the two organisms. The two bases different between ID and the other three MSP-la alleles are indicated by a single underline. In Fig. 11B "n" represents any deoxynucleotide. The FL, VA and WA alleles are identical from the transcription start site to the 5' end of the -35 region. The ID allele has a 1 base deletion within the -35 region, at position -30FL. The spacing between the -35 and -10 regions is maintained, however, by insertion of a T at position -22FL (see Figs. 10 and 11B) and all four alleles m tch that of the E. coli consensus sequence. Immediately 5' to the -35 region is an extremely A+T-rich stretch in which A or T occupy 23 of the 25 bases in that sequence. In the four alleles there is an apparently untranslated leader of 127 nucleotides for the FL, WA and VA isolates, or 71 nucleotides for the ID, as defined by the start of transcription at base 1FL and the start methioπine codon at bases 128FL-130FL Relative to FL, the VA and WA alleles are identical in this region except for an A to G transition at position 10FL. The ID allele, on the other hand, has a T to G transversion at position 8FL and deletions of 1, 51 and 4 bases in this region. Despite these differences in the 5' untranslated region the FL, WA and ID alleles are expressed at comparable levels in E. coli (DH5a). Although VA is not comparably expressed, this may be because of differences in plasmid copy number or products encoded by sequences 3' to the end of the MSP-la gene which are absent from the other recombinants. We have not pursued this question. We think that translation begins at the methionine codon of bases 128FL-130FL, for the following reasons: 1) The only long open reading frame in this gene begins just 5' to this codon coding; 2) although there is another methionine codon coding sequence upstream at bases 45FL-47FL, it is not in the same open reading frame as the long open reading frame and is absent altogether in the ID isolate; 3) monoclonal antibody 22B1 binds to a synthetic oligopeptide encoded by this wading frame (see Fig. 14); and 4) there are no other methionine codons in the open reading frame until a point beyond that contained in plasmid pAMTl, which expresses a fragment of the polypeptide. In each of the alleles one long open reading frame is present which extends to the same apparent stop codon sequence at bases 2429FL- 2431FL (see Fig. 10). Between alleles there is an extremely high degree of overall homology throughout the coding region, including a 639 bp region from bases 1686FL-2324FL that is completely conserved. However, there are three regions in the coding sequence with a high degree of variability. The first 30 bases of the DNA coding sequence comprises a hypervariable region wherein FL, VA and WA each have 4 substitutions, whereas ID has only 27 bases in the same region, of which 7 vary from the other isolates. The result is an associated N-terminal amino acid region shortened from 10 to 9 amino acids, with 4 substitutions between isolates, three of which are non-conservative. A similar clustering of substitutions at the 3' end results in 5 amino acid differences in the final 35 residues. Finally, the 120 bp stretch from bases 1184FL to 1202FL is a highly variable region, with 11 base substitutions resulting in the substitution of 11 out of 40 amino acids (see Figs. 10 and 12). Eight of these substitutions are non-conservative, and 7 of the 11 are in regions predicted to be short coil- turn structures. This may be important to host responses to this antigen. A notable feature of the MSP-la gene for all four isolates is the presence of a DNA tandem repeat region containing a series of similar tandemly repeated DNA sequences which each contain 84 or 87 bp. These DNA tandem repeat sequences code for the expression o polypeptide sequences having 28 or 29 amino acids, respectively. The tandemly repeated sequences are repeated two times in the VA isolate, four times in the WA isolate, six times in the ID isolate, and eight times in the FL isolate. It is interesting that each of the alleles varies by a multiplicative factor of two in the number of repeats but we cannot at this time ascribe any particular significance to this observation. The tandem repeats of 28 or 29 amino acid units immediately follo the N-terminal 10 (or 9) amino acids. Fig. 13 shows that the repeated amino acid sequences are presen in only five forms, herein termed repeat forms A-E, for all repea sequences contained in the tandem repeat regions of the four Anaplasm marginale isolates. Each geographical isolate allele contains two repea forms. The primary structures of the various repeat forms are highl conserved with 25 amino acids of the 28 or 29 mer sequences bein completely conserved in all five repeat forms defining all tandeml repeated sequences of these isolates. In each al 'e, the tandem repea domain begins or ends with a single copy of one repeat form wherea the second repeat form is present in one to seven copies. Variation in the number of tandem repeats present in each allele can completely explain the size polymorphisms of the Aml05U protein for these four geographical isolates of Anaplasma marginale without any need to invoke other mechanisms to explain the differences. The 28 and 29 mer amino acid sequences shown in Fig. 13 include conserved amino acid sequences DSSSA, GQQQESSVSSQS, EASTSS or QASTSS, and QLG. One or more of these sequences or their subunits can be significant in defining antigens in accordance with this invention. Antibody 22B1 selectively binds to sequences EASTSS and QASTSS as explained more fully below. Antibody titers have been developed in cattle against the Florida isolate 29 mer polypeptide shown in Fig. 13 as repeat form B. Coupling of one or more of these repeat sequences to additional polypeptide sequences may also be significant in stimulating an immune response which is characterized by the 28 or 29 mer amino acids sequences, or subunits thereof, such as the conserved subunits indicated in this document. These highly conserved tandem repeat units or homologous regions may also be conserved in other rickettsial organisms, thereby allowing additional rickettsial infections to be detected, treated or vaccinated against using the antigens and immunogens including these amino acid sequences, there subunits or combinations thereof. Analysis of the amino acid sequence information clearly indicates that the actual molecular weights of all antigenic proteins coded by the MSP-la genes of the different isolates is anomalous to the molecular weights predicted by sodium dodecylsulfate-polyacrylamide gel electrophoretic mobility comparisons with standards used in the testing. Each of the antigens migrates in electrophoresis in a manner appearing significantly larger than the encoded size. This variance between electrophoretic mobility and actual molecular weight is a recognized property of proteins containing domains of tandemly repeated amino acids. In addition to the various tandem repeat units, there are five other known sequence regions in the FL allele sharing significant homology with the tandem repeats. Four of these homologous regions form a series overlapping the same region within the repeat structure (exemplified by bases 236FL to 277FL). The first homologous region, bases 2240FL-2254FL, contains the DNA sequence GGTGGcCAGCAGCAg- (mismatches are in lower case, see Fig. 10). This sequence shares 13 of 15 bases with the homologous region of the tandem repeats. This region is within the long open reading frame, is in frame synchronization, and the base differences are silent (coding for the same amino acid), thus encoding the amino acid sequence GGQQQ in each region. The second homologous region (not shown in Fig. 10) is coded 2 by bases -1188FL to -1208FL which include the DNA sequence 2 TTAtGcGCaGATgcCaCcTCA. This region shares 14 of 21 bases. The 3 third homologous region, bases -1292FL to -1308FL (also not shown) 4 (TCAGOGGGTcGTCAGCA), shares 16 of 17. The fourth homologous 5 region, bases -1450FL to -1461FL (CgGCAGgAAGcG), shares 9 of 12 6 bases. The four homologous regions overlap an area of the repeat 7 sequence exemplified by bases 260FL-274FL, 236FL- 256FL- 254-270FL * and 266FL-277FL, respectively. The fifth homologous region, bases 9 -496FL to -516FL (CAGGaCcGcAaATGgGcCTCAA), shares 15 of 21 lo bases with a stretch exemplified by bases 302FL to 322FL. These u homologous regions may reflect the origin of the repeats as discussed 12 below. 13 Mapping the Neutralization-Sensitive Epitope- 14 In particular we wished to map the epitope recognized by 15 monoclonal 22B1 because of the demonstrated neutralization ability in 16 vitro and effectiveness of the antigen Aml05 (complex) and the binding 17 of monoclonal antibody 22B1 to this immunogen, thus indicating the 18 potential importance of this epitope in immune recognition. Plasmid 19 pAMTl encodes a subunit polypeptide of AmF105U which is recognized 20 by monoclonal antibody 22B1 yet contains only the N-termina. 10 amino 21 acid stretch and seven complete and one partial repeats. Because of 22 this we targeted our search to the repeat structure. Our approach was to assay the binding ability of monoclonal 22B1 to various synthetic oligopeptides containing overlapping stretches of the B repeat. The minimum structure necessary to bind this antibody was found to be the six amino acid sequence QASTSS. This sequence is present in one of two isoforms in each of the repeats, the alternative sequence being EASTSS. Both synthetic peptides bound equivalents amounts to monoclonal 22B1, whereas the 5 mer polypeptide. ASTSS and QASTS, did not bind antibody. The results of these experiments are summarized in Fig. 14. Oligopeptides of varying lengths and containing different regions of the A and B repeats of the antigen AmF105U polypeptide were assayed for monoclonal antibody 22B1 binding affinity. A (+) reaction indicates monoclonal 22B1 binding in these assays. An (-) reaction indicates no detectable monoclonal 22B1 binding. Results were obtained by solution- phase inhibition radioimmunoassay using 125 I-labeled AmF105, which were confirmed by an enzyme-linked immunosorbent assay and by immunoblot assays. The enzyme-linked assay was performed as described previously by Palmer et al., International Journal for Parasitology, 17, 1279 (1987), with the following modifications. The microtiter plates were first coated with bovine serum albumin to which was added the oligopeptides in 0.25% (w/v) glutaraldehyde, 10 mM sodium phosphate, 94 mM ethylenediamine tetraacetic acid, pH 6.8. Blocking and washing steps were done using a veronal buffer of the following composition: 145 mM sodium chloride, 1.8 mM sodium 5,5 ' '- diethylbarbiturate, 4.5 mM barbituric acid, 0.5mM MgCl 2 . The visualizing system was horseradish peroxidase coupled to recombinant Protein G. For the immunoblot assays, serial dilutions of the oligopeptides were spotted onto nitrocellulose previously coated with bovine serum albumin in water. The blots were fixed in 2 * 5% (v v) glutaraldehyde. After extensive washings with tris-buffered saline, the blots were incubated with monoclonal 22B1, then with rabbit anti-mouse IgG, and finally with 125 I- protein A, as described in Palmer it al., Science 231, 1299 (1986). Synthetic 29 mer oligopeptides containing the sequence shown in Fig. 13 as Form B were tested in calves to determine the antigenic capability of this sequence. Two calves were given 400 microgram doses at four times. The first injection was given in approximately 1-2 milliliters of Freund's complete adjuvant Three boosters containing similar amounts of the antigenic oligopeptide were given thereafter at approximately 2 week intervals. Three additional calves were similarly inoculated with another vaccine incorporating similar amounts of an antigen containing the synthetic 29 mer oligopeptides which had been polymerized with the cross-linking agent carbodiimide to produce antigens having approximate electrophoretic mobilities corresponding to molecular weights of 20,000 to 200,000 daltons, Science 144, 1344 (1964). All five of the calves were checked for serum antibody titers approximately 2 months after the initial inoculation and shortly after the last booster injection. Titers were analyzed against both immunoaffinity purified AmF105 and the 29 mer synthetic oligopeptide which were coated in the wells of the titer plates. The results of these test are shown below in Table 6. All calves were tested for titers prior to inoculation and found to have negligible reaction. « _______________________ TABLE 6 '- Titers AmF105 Titers 29 mer Oligo. 29 mer Oligopeptide Vaccine Calves 1 and 2 1:100 1:1000 29 mer Oligo. and Polymerized Carboiimide Vaccine Calf 3 1:10,000 1:10,000 Calves 4 and 5 1:1,000 1:10,000 Immunoaffi ty Purified AmF105 Vaccine Calf 6 1:10,000 1:1,000 __ mm ___ m _ m _ mm _ mmm __ m __________ t _ __ m ^^ The entire repeat domain is predicted by structural algorithms to be comprised of coil/turn segments, consistent with a short, linear epitope. However, the binding affinity of monoclonal 22B1 for the entire 29 mer repeat B was approximately two orders of magnitude greater than for either minimal epitope, suggesting some structural influence. Our results have revealed several interesting features of the MSP-la gene and its encoded polypeptide products including recombinant Aml05U, for the various isolates, and subunit antigens derivable therefrom. The presence of a tandem-repeat domain has not been reported before in a rickettsial surface protein, although they are found in the taxonomically distant streptococcal M proteins and in several eukaryotic parasite surface antigens. The variable numbers of repeats in this domain explains the extreme size polymorphisms of this polypeptide. The epitope bound by monoclonal antibody 22B1 was strictly conserved in each repeat of each isolate, even though it can function in neutralization of parasite infectivity. In addition to the variable numbers of repeats there are three highly variable regions in the polypeptide, including the N-terminal end. The gene uses promoter structures and spacings very similar t > the E. coli promoter consensus sequences. Despite the similarities between the MSP-la promoter and E. coli promoter consensus sequences, one significant difference emerged: No obvious ribosome binding site was detected in the untranslated leader region, even though this gene is expressed in E coli in appreciable amounts. The sequence GTGTGTG, found in the -11 to -5 (relative to the ATG codon) position may still base pair with the ribosomal RNA but with a lowered affinity. An unusual structural feature of the AmF105 polypeptide is that although it is a surface protein and accessible to antibody, no obvious signal sequence to promote its translocation to the outer membrane bilayer was detected. A hydropathy plot of the predicted polypeptide reveals five major hydrophobic stretches from amino acids 255FL-270FL, 541FL-557FL, 567FL-585FL, 631FL-650FL, and 662FL-678FL, the last four of which are sufficient in length and hydrophobicity to serve as transmembrane domains. One of these hydrophobic domains may serve as an uncleaved, internal signal sequence. The hypervariable nature of the N-terminal end of AmF105 suggests that this domain may not serve a structural or targeting function. On the other hand, this sequence and that of the highly variable region of amino acids 353FL to 392FL could serve immunologic functions, providing epitope(s) necessary for T-cell recognition. If so, T-cell and host subpopulations capable of responding to AmF105 could be modulated by these regions. Mutations in these regions therefore 2 may provide a level of antigen variation enabling survival of the parasite 2 in host populations immune to heterologous isolates. 3 The tandem repeat structures have been hypothesized to develop 4 by multiple events of unequal homologous recombination and/or slipped- 5 strand mispairing. The origin of the tandem repeats in AmF105 is 6 unknown. Sequences sharing significant homology with the repeats are 7 also seen at other sites within and outside the MSP-la gene coding 8 sequence. Given the lengths of the tandem repeats (84 or 87 bp), 9 unequal homologous recombination is the more likely mechanism as 2i? longer sequences reduce the probability of slipped-strand mispairing. u Our characterization of multiple MSP-la gene alleles has clarified 12 one molecular basis for rickettsial surface antigen size polymorphisms. 13 Having defined several features of the MSP-la gene will enable us to 14 map T-cell epitopes of AmF105 and to assess the potential for T-cell 15 epitope-based antigen variation in Anaplasma marginale. The applicability 16 to other rickettsial organisms can also be further investigated. Definition 17 of a conserved neutralization-sensitive epitope allows us to further assess 18 the immunoprotective of synthetic, including recombinantly produced, 19 peptide-based vaccines. 20 In addition to the four alleles of the MSP-la gene, we have 21 cloned the Florida isolate MSP-lb gene using procedures the same as 22 or very similar to those described herein. The MSP-lb gene for the Florida isolate codes for the production of recombinant AmF105L, the second surface protein subunit of the MSP-1 complex in the Florida isolate of Anaplasma marginale. Fig. 15 is a restriction enzyme map for the MSP-lb Florida gene showing the cutting sites for a variety of enzymes. Fig. 16 shows the DNA and associated amino acid sequences coded for by the AmF105L gene. The last part of Fig. 16 also shows the amino acid composition of the expressed recombinant antigen and the calculated molecular weight of 80,359.85 daltons which compares to the electrophoretic mobility measurements of approximately 100-105 kilodaltons. Thus the production of recombinant MSP-1 complexes can be developed by co-expression of the MSP-la and MSP-lb genes in a heterologous system. The amino acid sequence abbreviations used in this document are shown in the Appendix A filed herewith. THE TWENTY AMINO ACIDS 3-Letter 1 -Letter Type of Amino Acid Symbol Symbol Hydrophobic (Aliphatic Side Chain) Glycine Gly G Alaπiπe Aia A Valine Val V Leucine Leu L Isoleucine He 1 Basic (Diamino) Arginiπe Arg R Lysine Lvs K Acidic (Dicarboxylic) Glutamic acid GIu E Aspartic acid Asp D Amide-Containing Glutamine Gin Q Asparagine Asn N Hydroxyl-Containing Threoniπe Thr T Serine Ser s Sulfur-Con ta in ing Cysteine Cys c Methionine Met M Aromatic Phenylaiaπine Phe F Tyros ine Tyr Y Heterocyclic Tryptophan Tφ W Proline Pro P Histidine His H APPENDIX A

Claims: 2 CLAIMS 2 1. A purified antigenic peptide containing at least one amino 3 acid sequence selected from a group consisting of the following amino 4 acid sequences: 5 glutamic a d-alanine-serine-threonine-serine-serine; and 6 glutarmne-alanine-ser-ne-threonine-serine-serine. 7 * 2. A purified antigenic peptide according to claim 1 wherein 9 said antigenic peptide is produced by a cell which includes recombinant lo DNA coding for the production of said antigenic peptide. u 12 3. A purified antigenic peptide according to claim 1 wherein 13 said antigenic peptide is produced by artificial peptide synthesis. 14 15 4. A purified antigenic peptide according to claim 1 wherein 16 said antigenic peptide is immunogenic to cause resistance to infection by 17 at least one species of rickettsial organism. 18 19 5. A purified antigenic peptide according to claim 1 wherein 20 said antigenic peptide is immunogenic to cause resistance to infection by 21 at least one rickettsial organism of the genus Anaplasma. 22 2 6. A purified antigenic peptide according to claim 1 wherein 2 said antigenic peptide is immunogenic to cause resistance to infection by 3 at least Anaplasma marginale. 4 5 7. A purified antigenic peptide according to claim 1 wherein 6 said antigenic peptide is immunogenic in mammals to cause resistance 7 to infection by at least Anaplasma marginale. 8 9 8. A purified antigenic peptide according to claim 1 wherein 10 said antigemc peptide has a molecular weight of at least 15 kilodaltons. ll 12 9. A purified antigenic peptide according to claim 1 wherein 13 said antigenic peptide has relative mobility in gel electrophoresis which 14 corresponds to an approximate molecular weight of at least 15 15 kilodaltons. 16 17 10. A purified antigenic peptide according to claim 1 and further 18 defined to include an amino acid sequence comprising the sequence 19 described as Form A of Fig. 13. 20 11. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising the sequence described as Form B of Fig. 13. 12. A purified antigenic peptide according to claim 1 and further defined to include an -min ~ add sequence comprising the sequence described as Form C of Fig. 13. 13. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising the sequence described as Form D of Fig. 13. 14. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising the sequence described as Form E of Fig. 13. 15. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising glutamine-leucine- glycine. 16. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising aspartic acid- serine-serine-serine-alanine. 17. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising glycine-glycine- glutaniine-glutamine-glutamine. 18. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising serine-glycine- glutamine-glutamine-glutamine. 19. A purified antigenic peptide according to claim 1 and further defined to include an amino acid sequence comprising glycine-glutamine- glutamine-glutamine-glutamic acid-serine-seriπe-valine-serine-serine-glutamine- serine. 20. A purified antigenic peptide according to claim 1 and further defined to include at least two amino acid sequences selected from a group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamme-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutanaine; and glycine-glutamine-glutam e-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 21. A purified antigenic peptide according to claim 1 and further defined to include at least two tandem repeat amino acid sequences which include an amino acid sequence comprising glutamic acid-alanine- serine-threoπine-serine-serine. 22. A purified antigenic peptide according to claim 1 and further defined to include at least two tandem repeat amino acid sequences which include a first amino add sequence comprising glutamic acid- alanine-serine-threonine-seriπe-serine, and a second amino acid sequence comprising glutamine-leucine-glycine. 23. A purified antigenic peptide according to claim 1 and further defined to include at least two tandem repeat amino acid sequences which include a first amino acid sequence comprising glutamic acid- alanine-serine-threonine-serine-serine, and a second amino acid sequence comprising aspartic acid-serine-serine-serine-alanine. 2 24. A purified antigenic peptide according to claim 1 and further 2 defined to include at least two tandem repeat amino acid sequences 3 which include a first amino acid sequence comprising glutamic acid- 4 alanine-serine-threomne-serine-serine, and a second amino acid sequence 5 comprising glycine-glycine-glutamine-glutamine-glutamine. 6 7 25. A purified antigenic peptide according to claim 1 and further 8 defined to include at least two tandem repeat amino acid sequences which include a first amino acid sequence comprising glutamic acid¬ ic alanine-serine-threonine-serine-serine, and a second amino acid sequence u comprising serine-glycine-glutamine-glutamine-glutamine. 12 13 26. A purified antigenic peptide according to claim 1 and further 14 defined to include at least two tandem repeat amino acid sequences 15 which include a first amino acid sequence comprising glutamic acid- 16 alaπine-serine-threonine-serine-serine, and a second amino acid sequence 17 comprising glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine 18 valiπe-serine-serine-glutamine-serine. 19 20 27. A purified antigenic peptide according to claim 1 and furthe 21 defined to include at least two tandem repeat amino acid sequence 22 which include a first amino acid sequence comprising glutamic acid alanine-serine-tltreonine-serine-serine, and at least second and third amino acid sequences; said second and third amino acid sequences being selected from the group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic- acid-serine-serine-serine-alanine; glydne-glycine-glutamine-glutamine-glutamine; sέirine-glycine-glutaπiine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 28. A purified antigenic peptide according to claim 1 and further defined to include at least two tandem repeat amino acid sequences which include the following amino acid sequences: glutamic add-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-seriπe-serine-serine-alaπine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valiπe- serine-serine-glutamine-serine. 29. A purified antigenic peptide which is immunogenic to provide resistence to infection by at least one rickettsial organism containing at 2 least one amino acid sequence selected from a group consisting of the 2 following amino acid sequences: 3 glutamic acid-alanme-serine-threonine-serine-serine; and 4 glutaniine-alanine-serine-tiireonine-serine-serine. 5 6 30. A purified antigenic peptide according to claim 29 and 7 further defined to include an amino add sequence comprising glutamine- * leucine-glycine. 9 lo 31. A purified antigenic peptide according to claim 29 and u further defined to include an amino acid sequence comprising aspartic 12 add-serine-serine-serine-alanine. 13 14 32. A purified antigenic peptide according to claim 29 and is further defined to include an amino acid sequence comprising glycine- 16 glycine-glutamine-glutamine-glutamine. 17 18 33. A purified antigenic peptide according to claim 29 and 19 further defined to include an amino acid sequence comprising serine- 20 glycine-gluti_._nine-glutam.ne -glutamine. 21 34. A purified antigenic peptide according to claim 29 and further defined to include an amino acid sequence comprising glycine- glutamine-glutamine-glutainine-glutamic acid-serine-serine-valine-serine-serine- glutamine-serine. 35. A purified antigenic peptide according to claim 29 and further defined to include at least two amino acid sequences from a group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic add-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutantine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutεmic acid-serine-serine-valine- serine-serine-glutamine-serine. 36. A purified antigenic peptide according to claim 29 and further defined to include at least two tandem repeat amino acid sequences which include at least one amino acid sequence selected from said group. 37. A purified antigenic peptide according to claim 29 and further defined to include at least two tandem repeat amino acid sequences which include a first amino add sequence comprising glutami acid-alanine-serine-threoπine-serine-serine, and a second amino aci sequence comprising glutamine-leucine-glyciπe. 38. A purified antigenic peptide according to claim 29 an further defined to include at least two tandem repeat amino aci sequences which include a first amino add sequence comprising glutami add-alanine-serine-threonine-serine-serine, and a second amino aci sequence comprising aspartic acid-serine-serine-serine-alaπine. 39. A purified antigenic peptide according to claim 29 an further defined to include at least two tandem repeat amino aci sequences which include a first amino acid sequence comprising glutami acid-alaniπe-serine-threonine-serine-serine and a second amino aci sequence comprising glycine-glycine-glutamine-glutamine-glutamine. 40. A purified antigenic peptide according to claim 29 an further defined to include at least two tandem repeat amino aci sequences which include a first amino acid sequence comprising glutam acid-alanine-serine-threonine-serine-serine and a second amino ac sequence comprising serine-glycine-glutamine-glutamine-glutamine. 41. A purified antigenic peptide according to claim 29 and further defined to include at least two tandem repeat amino acid sequences which include a first amino add sequence comprising glutamic add-alanme-serine-threonine-serine-serine and a second amino acid sequence comprising glycme-glutamme-glutamine-glutamiπe-glutamic acid- serine-serine-valine-serine-serine-glutamine-serine. 42. A purified antigenic peptide according to claim 29 and further defined to include at least two tandem repeat amino acid sequences which include a first amino acid sequence comprising glutamic add-alanine-serine-threonine-serine-serine and at least second and third amino add sequences; said second and third amino acid sequences being from the group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic add-serine-serine-serine-alanine; glycine-glyciπe-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 43. A purified antigenic peptide according to claim 29 and further defined to include at least two tandem repeat amino acid sequences which include the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 44. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including a first repeated amino acid sequence and a second repeated amino acid sequence; said first repeated amino acid sequence being selected from a first group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; and glutamine-alanine-serine-threonine-serine-serine; said second repeated amino acid sequence being selected from a second group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamiπe-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamme-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 45. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two tandem repeat amino acid sequences; said two tandem repeat amino add sequences each including at least one repeated amino add sequence selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threoπine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glyciπe-glutamine-glutamine-glutamine; serine-glycine-giutamine-glutamine-glutamine; and glycine-glutamine-glutamiπe-glutamine-glutamic acid-serine-serine-valine- serine-seri._e-glutamine-serine. 46. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including at least two repeated amino acid sequences selected from a group consisting of the following amino add sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutømine-glutamine-glutamine; and glycine-glutaπi e-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 47. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including at least three repeated amino acid sequences selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutarnine-glutamine-glutamine; serine-glycine-glutamine-glutarnine-glutamine; and glycine-glutainine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 48. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including at least four repeated amino acid sequences selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 49. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence approximately as specified in Fig. 12 for the Florida isolate. 50. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence approximately as specified in Fig. 12 for the Virginia isolate. 51. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence approximately as specified in Fig. 12 for the Washington isolate. 52. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence approximately as specified in Fig. 12 for the Idaho isolate. 53. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least two amino acid sequences selected from a group consisting of the following amino acid sequences: 2 glutamic acid-alanine-serine-threonine-serine-serine; 2 glutamine-leucine-glycine; 3 aspartic acid-seriπe-serine-serine-alanine; 4 glycine-glycine-glutainine-glutamine-glutamine; 5 serine-glycine-glutamine-glutamine-glutamine; and 6 glycine-glutainine-glutamine-glutamine-gluta ic acid-serine-serine-valine- 7 serine-serine-glutamine-serine. 8 9 54. A purified antigenic peptide according to claim 53 and 10 further characterized by an ability to bind with immune serum of an u animal given a rickettsial organism against which immunogenic resistance 12 to infection is desired. 13 14 55. A purified antigenic peptide which is immunogenic to provide 15 resistance to infection by at least one rickettsial organism containing an 16 amino acid sequence comprising at least three amino acid sequences 17 selected from a group consisting of the following amino add sequences: 18 glutamic acid-alamne-serine-threoπine-serine-serine; 19 glutamine-leucine-glycine; 2. aspartic acid-serine-seriπe-serine-alanine; 21 glycine-glycine-glutamine-glutamine-glutamine; 22 serine-glycine-glutamine-glutamiπe-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine- vahne-serine-serine-glutamine-serine. 56. A purified antigenic peptide according to claim 55 and further characterized by an ability to bind with immune serum of an animal given a rickettsial organism against which immunogenic resistance to infection is desired. 51. A purified antigenic peptide which is immunogenic to provide resistance to infection by at least one rickettsial organism containing an amino acid sequence comprising at least four amino acid sequences selected from a group consisting of the following amino add sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamiπe-glutamiπe; and giycine-glutamiπe-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 58. A purified antigenic peptide according to claim 57 and further characterized by an ability to bind with immune serum of an animal given a rickettsial organism against which immunogenic resistance to infection is desired. 59. A vaccine for inducing an immune response in an animal susceptible to infection by a rickettsia which is protective to reduce the severity or prevent infection by said rickettsia, comprising: at least one purified antigenic peptide as defined in claims 24, 30, 31, 37, 38, 39 or 40. 60. A vacdne for inducing an immune response in an animal susceptible to infection by a rickettsia of the genus Anaplasma which is protective to reduce the severity or prevent infection by said rickettsia, comprising at least one purified antigenic peptide containing at least one amino acid sequence selected from a group consisting of the following amino acid sequences: glutamic add-alanine-serine-threoπine-seriπe-seriπe; glutamine-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic add-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutaiπine-serine. 61. A vaccine according to claim 60 and further characterized by being adapted for indudng an immune response to at least one rickettsia of the species Anaplasma marginale. 62. A vaccine for inducing an immune response in an animal susceptible to infection by Anaplasma marginale which is protective to reduce the severity or prevent infection thereby, comprising at least one purified antigenic peptide containing at least one amino acid sequence selected from a group consisting of the following amino add sequences: glutamic acid-alaπine-serine-threonine-serine-serine; and glutamine-alanine-serine-threonine-serine-seriπe. 63. A vaccine according to claim 62 wherein said antigeni peptide is produced by a cell which includes recombinant nucleic aci coding for the production of said antigenic peptide. 64. A vaccine according to claim 62 wherein said antigeni peptide is produced by artificial peptide synthesis. 65. A vaccine according to claim 62 wherein said antigenic peptide and further comprises at least one additional amino acid sequence selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamme-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serme-alanine; glycine-glyciπe-glutamine-glutamine-glutamine; sei±ie-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutaniine-glutarnine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 66. A vaccine according to claim 65 wherein said vaccine is adapted to immunize mammals. 61. A vaccine according to claim 62 wherein said antigenic peptide is further defined to include at least two tandem repeat amino add sequences which include at least one amino acid sequence selected from said group. 68. A vaccine according to claim 62 wherein said antigenic peptide is further defined to include at least two tandem repeat amino add sequences which include at least one amino acid sequence selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutaniine-alanine-serine-threonine-serine-serine; glutamine-leudne-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; seriπe-glycine-glutamine-glutamine-glutamine; and glycine-glutaπiine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 69. A vaccine according to claim 62 wherein said antigenic peptide is further defined to include at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including a first repeated amino acid sequence and a second repeated amino acid sequence; said first repeated amino acid sequence being selected from a first group consisting of the following amino acid sequ .nces: glutamic acid-alanine-serine-threonine-serine-serine; and glutamine-alanine-serine-threonine-serine-serine; said second repeated amino add sequence being selected from a second group consisting of the following amino acid sequences: glutamine-leucine-glycine; aspartic add-serine-serine-serine-alanine; glycine-glycine-glutamme-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamme-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 70. A vaccine for inducing an immune response in an animal susceptible to infection by Anaplasma marginale which is protective to reduce the severity or prevent infection thereby, comprising at least one purified antigenic peptide having at least two tandem repeat amino acid sequences; said two tande repeat amino acid sequences each including at least two repeated amino acid sequences selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alaπine; glycine-glycine-glrtar..ine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 71. A vaccine for inducing an immune response in an animal susceptible to infection by Anaplasma marginale which is protective to reduce the severity or prevent infection thereby, comprising at least one purified antigenic peptide having at least two tandem repeat amino acid sequences; said two tandem repeat amino acid sequences each including at least three repeated amino acid sequences selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serme-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alaniπe; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 72. A vaccine for inducing an immune response in an animal susceptible to infection by Anaplasma. marginale which is protective to reduce the severity or prevent infection thereby, comprising at least one purified antigenic peptide having at least two tandem repeat amino acid -120- 2 sequences; said two tandem repeat amino acid sequences each including 2 at least four repeated amino acid sequences selected from a group 3 consisting of the following amino acid sequences: 4 glutamic acid-alamne-serine-threonine-serine-serine; 5 glutamine-leucine-glycine; 6 aspartic acid-serine-serine-serine-alanine; 7 glycine-glycine-glutamme-glutamme-glutamine; 8 serine-glycine-glutamine-glutamine-glutamine; and 9 glycine-glutamine-glutaπiine-glutamine-glutamic acid-serine-serine-valine- 10 serine-serine-glutamine-serine. ll 12 73. A method for inducing an immune response in an animal 13 susceptible to infection by at least one rickettsial organism to provide 14 at least some protection to reduce or prevent infection by said at least 15 one rickettsial organism,, comprising: 16 inoculating the animal with an immunogenic antigen in an amount 17 sufficient to induce an immune response; said immunogenic antigen 18 including at least one purified peptide containing at least one amino 19 acid sequence selected from a group consisting of the following amino 20 acid sequences: 21 glutamic add-alanine-serine-threonine-serine-serine; 22 glutamine-alanine-serine-threoπine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamiπe; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamme-glutaπiine-glutamine-glutamic add-serine-serine-valine- serine-serine-glutamine-serine. 74. A method according to claim 73 wherein said immunogenic antigen includes at least one purified peptide which includes at least one amino add sequence comprising glutamic add-alanine-serine-threonine- serine-serine. 15. A method according to claim 73 wherein said immunogenic antigen includes at least one purified peptide which includes at least one amino acid sequence comprising glutamine-alanine-serine-threonine- serine-serine. 16. A method according to claim 73 wherein said immunogenic antigen includes at least one purified peptide which includes at least two tandem repeat amino acid sequences; at least two of said tander repeat amino acid sequences containing at least one amino acid 2 sequence selected from a group consisting of the following amino acid 2 sequences: 3 glutamic acid-alanine-serine-threonine-serine-serine; 4 glutemine-alanine-serine-threoriine-serine-serine; 5 glutamine-leucine-glycine; 6 aspartic acid-serine-serine-serine-alanine; 7 glydne-glycine-glutamme-glutamine-glutamiπe; 8 serine-glycine-glutamine-glutamine-glutamine; and 9 gtycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- lo serine-serine-glutamine-serine. ll 12 11. A diagnostic test for detecting antibody against a rickettsial 13 parasite including at least one purified antigenic peptide containing at 14 least one amino acid sequence selected from a group consisting of the is following amino acid sequences: 16 glutamic add-alanine-serine-threonine-serine-serine; and 17 glutamine-alanine-serine-threonine-serine-serine. 18 19 78. A diagnostic test according to claim 77 wherein at least one 20 amino acid sequence selected from said group is produced from a cell 21 which includes recombinant nucleic acid which codes for the production 22 of said amino acid sequence. 79. A diagnostic test according to claim 77 wherein at least one amino acid sequence selected from said group is produced by artificial peptide synthesis. 80. A diagnostic test according to claim 77 wherein the test is adapted to detect antibody raised against a rickettsial parasite of the genus Anaplasma. 81. A diagnostic test according to claim 77 wherein the test is adapted to detect antibody raised against a rickettsial parasite of the spedes Anaplasma marginale. 82. Recombinant nucleic acid coding for the expression of at least one antigenic peptide capable of inducing an immune response to a rickettsial parasite which is protective to reduce the severity or prevent infection by said rickettsial parasite. 83. The recombinant nucleic acid of claim 82 further defined as deoxyribonucleic acid. 84. The recombinant nucleic add of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide capable of inducing an immune response to a rickettsial parasite of the genus Anaplasma. 85. The recombinant nucleic add of claim 84 further defined as deoxyribonudeic add. 86. The recombinant nucleic add of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide capable of inducing an immune response to a rickettsial parasite of the species Anaplasma marginale. 87. The recombinant nucleic add of claim 86, 94, 95, 96 further defined as deoxyribonudeic acid. 88. The recombinant nucleic acid of claim 82 further defined as nucleic add coding for the expression of at least one antigenic peptide including the amino acid sequence comprising glutamic acid-alanine-serine- thi onine-serine-serine. 89. The recombinant nucleic acid of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide including the amino acid sequence comprising glutamine-alanine-serine- threonine-serine-serine. 90. The recombinant nucleic add of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide including at least one amino acid sequence selected from a group consisting of the following amino add sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutaπiine-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valiπe serine-serine-glutamine-serine. 91. The recombinant nucleic acid of claim 82 further defined a nucleic acid codir. for the expression of at least one antigenic peptid including at least two amino acid sequences selected from a grou consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; gluta-nine-alaπine-ser e-tlireoniπe-serine-serine; glutamine-leucine-glycine; aspartic add-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutaπiine-glutamme-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 92. The recombinant nucleic add of claim 82 further defined as nucleic add coding for the expression of at least one antigenic peptide including at least three amino acid sequences selected from a group consisting of the following amino acid sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glydne-glydne-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glut_.mine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 93. The recombinant nucleic acid of claim 82 further defined a nucleic acid coding for the expression of at least one antigenic peptid including at least four amino acid sequences selected from a grou consisting of the following amino add sequences: glutamic acid-alanine-serine-threonine-serine-serine; glutamine-alanine-serine-threoπiπe-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valiπe serine-serine-glutamine-serine. 94. The recombinant nucleic acid of claim 82 further defined nucleic acid coding for the expression of at least one antigemc peptid capable of inducing an immune response to a rickettsial parasite of th species Anaplasma marginale; and further characterized by said antigen peptide including at least one amino acid sequence selected from group consisting of the following amino acid sequences: glutamic acid-alanine-seriπe-threonine-serine-serine, glutamine-alaniπe-seriπe-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamme-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 95. The recombinant nucleic add of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide capable of inducing an immune response to a rickettsial parasite of the species Anaplasma marginale; and further characterized by said antigenic peptide including at least two tandem repeat amino acid sequences which contain at least one amino acid sequence selected from a group consisting of the following amino acid sequences: glutamic add-alanine-serine-threonine-serine-serine; glutamine-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamiπe-glutamine-glutamic acid-serine-serine-vαline- serine-serine-glutamine-serine. 96. The recombinant nucleic add of claim 82 further defined as nucleic acid coding for the expression of at least one antigenic peptide capable of inducing an immune response to a rickettsial parasite of the species Anaplasma marginale; and further characterized by said antigenic peptide including at least two tandem repeat amino acid sequences which contain the amino acid sequence glutamic acid-alanine-serine-threonine- serine-serine and at least one amino acid sequence selected from a group consisting of the following amino acid sequences: glutamine-alanine-serine-threonine-serine-serine; glutamine-leucine-glycine; aspartic acid-serine-serine-serine-alanine; glycine-glycine-glutamine-glutamine-glutamine; serine-glycine-glutamine-glutamine-glutamine; and glycine-glutamine-glutamine-glutamine-glutamic acid-serine-serine-valine- serine-serine-glutamine-serine. 97. A substantially pure antigenic surface protein of Anaplasma marginale an active fragment thereof, or an immunologically similar protein produced by polypeptide synthesis or genetic engineering which, when inoculated into a ruminant cow, is capable of inducing an immune response in said ruminant to Anaplasma marginale. 98. A nucleic acid probe for detecting native DNA of a rickettsial parasite including at least one nucleic add sequence selected from a group consisting of AGTGGTCAGCAGCAA and GGTGGTCAGCAGCAA.

Inventors: ALLRED DAVID REDDING, BARBET ANTHONY FRANCIS, DAVIS WILLIAM CHARLES, MCGUIRE TRAVIS CLINTON, PALMER GUY HUGHES, ALLRED, DAVID, REDDING, BARBET, ANTHONY, FRANCIS, DAVIS, WILLIAM, CHARLES, MCGUIRE, TRAVIS, CLINTON, PALMER, GUY, HUGHES
Applicants: UNIV WASHINGTON, WASHINGTON STATE UNIVERSITY RESEARCH FOUNDATION, I
Application year: 1990
Main classification: A61K39
IPC codes: A61K39, C07K14, C12N5, C12N15, C12P21, C12Q1, C12R1, G01N33