WO1992003156A1

WO1992003156A1 - Membrane-derived antiflea vaccines

Info

Publication number: WO1992003156A1
Application number: PCT/US1991/005852
Authority: WO
Inventors: Joan P. Opdebeeck; Robyn E. Boreham; Ann E. Arfsten; Andrew W. Heath; Miles K. Yamanake; Lynnor B. Stevenson; Beverly Dale
Original assignee: University of Queensland UQ; Paravax Inc
Current assignee: University of Queensland UQ; Paravax Inc
Priority date: 1990-08-22
Filing date: 1991-08-15
Publication date: 1992-03-05
Anticipated expiration: 1993-02-22
Also published as: AU8537091A

Abstract

A vaccine for protecting avian and mammalian subjects against flea infestation comprises the membranes or membrane extracts of fleas, or the antigenic components thereof. This also has the effect of reducing flea populations in the environment of the subject. Antibodies raised by these vaccines are also useful in purification and diagnosis. Especially preferred are the membranes and their components derived from flea midguts.

Description

MEMBRANE-DERIVED ANTIFLEA VACCINES

Technical Field

The invention relates to prevention of flea infestation in mammals and birds. More particularly, the invention concerns vaccines derived from membranous elements of fleas which are used to immunize mammalian and avian subjects against flea infestation.

Background Art Fleas are insects which behave as ectoparasites for birds and mammals. They are a serious nuisance both in the raising of animals which are sources of food and fiber and in the nurture of pet cats and dogs. The problem in the latter situation is particularly serious because the infestation also becomes a source of annoyance for the pet owner who may find his or her home generally contaminated with fleas which feed on the pets, and these parasites can induce an allergic reaction in both the pets and humans. The prevalence of flea aller- gic dermatitis (FAD) constitutes the foremost veterinary der atological problem in the U.S. (Kwochka, K. . , Vet Clin North Am (1987) 12:1235-1262). Furthermore, the life cycle of the flea favors a survival of intermediate stages since the adult flea feeds and copulates frequently and contaminates the entire environment with eggs (Nesbitt, G.H., et al. , J Am Vet Med Assoc (1978) 173;282-288; Soulsby, E.J.L. , in Helminths. Arthropods and Protozoa of Domesticated Animals, 7th ed. (1982) , Lea and Febiger, eds., Philadelphia, PA, pages 378-384). Although flea (i.e., insect) parasitism can be distinguished from that associated with other parasites, such as helminths, which are worms, and ticks, which are arachnids, control of all forms of parasitism has generally involved internal or external applications of chemicals. Commonly encountered approaches to control the flea problem are generally focused on use of insecti¬ cides in formulations such as sprays, shampoos, dusts, dips, or foams, or in pet collars. None are notably successful. While some of these products are effica¬ cious, they are often not successful in reducing flea populations on the pet or in the home for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required) , (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration, and (3) the emergence of flea populations resistant to the prescribed dose of pesticide. In addition, some flea-control clients are adverse to the use of certain chemicals in their home or on their pet that will remain as residual contaminants in the environment.

Efforts to find nontoxic approaches to flea control have resulted in the recent introduction of insect growth regulators (IGRs) such as methoprene which mimic flea hormones and affect flea larval development. A vaccine to reduce flea infestation on the pet and in the home would constitute another nonchemical approach to flea control and would avoid many of the compliance issues necessary for correct pesticide or IGR administration.

A number of attempts have been made to provide a vaccination approach to endoparasites, to other ecto¬ parasites, or to ectoparasites in general. Danish pat¬ ent 2644149 (1978) suggests the use of antigenic extracts from intermediate hosts in order to prepare antiparasitic vaccines. The focus of this work is with respect to Schistosomes. PCT application WO88/01277 to Australian National University discloses recombinant DNA encoding a helminth parasite antigen and suggests its use in constructing vaccines. PCT application W087/05513 (US 4,814,170) assigned to Apht&n Corporation describes antiendo- or antiectoparasite vaccines in general, which are derived from endocrine products, such as juvenile hormones. PCT application WO86/02839 suggests vaccines against helminths which contain suspensions, homogenates or extracts of nonparasitic nematode species. Presuma¬ bly, these nonparasitic species are closely enough related to the parasitic forms to engender appropriate antibodies. A similar approach with respect to protozoa is disclosed in PCT application WO83/03199. British application 1580539A published in 1980 suggests an antiparasitic vaccine derived from secretions of the parasite. None of these approaches are specifically directed to protection against flea infestation.

The problem of flea allergic dermatitis (FAD) has, however, inspired considerable study of the immuno- logical response of hosts to flea antigens which are, presumably, made available for host exposure through the saliva. These studies focus on the nature of the immune response in the host to these antigens, which do not necessarily result in protection of the host against infestation (Halliwell, R.E.W., J Immunol (1973) 110:442-430; Halliwell, R.E.W., et al., J Allerσ Clin Immunol (1978) £2.:236-2 2; Halliwell, R.E.W., et al.. Vet Immunol Immunopathol (1985) 8.:215-223; Wikel, S.K., Vet Parasitol (1984) 11:321-329). An early study which used FAD as a criterion for response to flea antigens, conducted in 1939, suggested that the FAD response, however, might somehow protect the host in such a way so as to prevent flea bites (Cherney, L.S., et al.. Am J Trop Med (1939) 19.:327-332) .

Others have suggested this general concept with regard to other parasites such as ticks and mosquitoes where it is considered that the hypersensitivity genera¬ ted by salivary antigens may provide some protection against feeding of the parasite (Ribeiro, J.M.C., Ann Rev Entomol (1987) 3_2.:463-478; Brown, S.J., Vet Parasitol (1988) 29_:235-264; Wikel, S.K., Vet Parasitol (1988): 2_9:235-264. Since one of the features needed in an effective antiflea vaccine, especially in the context of protecting pets against flea infestation, is the incen¬ tive for owner compliance, it is clearly undesirable for any vaccine to behave in such a way as to correlate efficacy with hypersensitivity and dermatitis.

An alternate concept, that of using "hidden" antigens, has been extensively studied for defense against ticks, e.g., Boophilus microplus (Opdebeeck, J.P. , et al., Immunol (1988) 6_:363-367; Opdebeeck, J.P., et al.. Parasite Immunol (1988) .10:405-410; Wong, J.Y.M. and Opdebeeck, J.P., Immunol (1989) j56.:149; Opdebeeck, J.P., et al., Immunol (1989) .67:388; European patent application 208,507, published 14 January 1987. See also Willadsen, P., et al. , J Immunol (1989) 143:1346-1351; Rand, K.N., et al., Proc Natl Acad Sci (USA) (1989)

8 :9657-9661; pcT application W088/3929, published 2 June 1988; Kemp, D.H. , et al. , Int J Parasitol (1986) 16:115- 120. This approach is also reviewed by Wikel, S.K. , Vet Parasitol (1988) (supra) ) . This approach has also been attempted using the thoracic muscles of the stable fly

(Schlein, Y. , et al. , Physiolog Entomol (1976) 1:55-59). Furthermore, it has been shown that IgG components from the host can be found intact in the body cavity of the parasitic insect which indicates the passage of an immunoglobulin through the midgut wall (Chinzei, Y., et al., Med Vet Entomol (1987) 1:409-416; Hatfield, P.R., Med Vet Entomol (1988) 2:339-345; Vaughan, J.A. , et al., J Med Entomol (1988) 25.:472-474) . While this phenomenon of immunoglobulin passage through the gut wall has also been shown in fleas, the work related not to antibodies to flea antigens, but rather to antibodies formed to rickettsia organisms carried in the flea (Azad, A.F., et al.. Am J Trop Med Hvg (1987) 37:629-635) .

More recently, a thesis prepared in support of the award of a Ph.D. degree at the University of London by Hatfield, P.R. (1986) investigated the immunization of hosts with homogenates or crude extracts of mosquitoes and fleas. The study showed that fleas that fed on mice immunized with flea homogenates and that ingested flea- specific antibodies showed a significant increase in mortality. Similar results were obtained for mosquitoes. The flea aspect of the study used X. Cheopis and utilized a solubilized extract of whole body homogenates. Only the soluble extract was employed. The present invention, in contrast, utilizes membrane associated flea antigens, preferably from the midgut. The use of these hidden antigens provides a nonallergic vaccine which will elicit antibodies directly reactive with the internal organs of the infesting insects.

Disclosure of the Invention

The invention provides a vaccine useful to immunize mammalian and avian subjects in such a manner as to lower infestation by the insect ectoparasites which are responsible for the problems associated with extensive flea infestation, and to lower the population of fleas in the surrounding environment. The vaccine employs antigens which are associated with membranes of the major parasitic fleas of dogs and cats, e.g.. Ctenoceoha1ides felis. and provides nonallergic protection against this nuisance.

Therefore, in one aspect, the invention is directed to a vaccine for protection of a subject against infestation by fleas, which vaccine comprises an amount of flea membrane or flea membrane extract, or an effec¬ tive antigenic component of said membrane or extract, which is effective to confer resistance to the infesta¬ tion, either by killing the insects or by incapacitating them in some way, or both. The successful application of the vaccine also lowers the flea population in the animal's surroundings. The invention is also directed to antibodies specifically immunoreactive with these anti¬ gens. In an additional aspect, the invention is directed to a method to protect a subject against infestation by fleas and to reduce the flea population in its environ¬ ment which comprises administering the vaccine of the invention. In still another aspect, the invention is directed to methods to prepare the effective vaccines.

Brief Description of the Drawings

Figure 1 shows a diagram of flea anatomy indi¬ cating the internal organs of interest.

Figure 2 is a halftone copy of a photograph showing the results of an SDS gel run on a total flea membrane and total flea supernatant antigens prepared as described in Example 1A.

Figure 3 is a halftone copy of a photograph showing the results of an SDS gel run on a total flea membrane and total flea supernatant antigens prepared as described in Example IB.

Figure 4 is a halftone copy of a photograph showing the results of an SDS gel run on a gut flea membrane antigen preparation. Modes of Carrying Out the Invention

The invention vaccines are preparations which are or contain antigens associated with internal flea membranes. The membranes may be obtained as total membrane preparations, or the insect may be dissected and an individual membrane type^~ used as a source for the antigen used in the vaccine. The vaccines are formulated in conventional ways, optionally using adjuvants either in or along with the formulation. Low molecular weight antigens are also conjugated to carriers or to themselves if necessary to enhance immunogenicity.

Preparation of the Membrane-Associated Flea Antigens Large numbers of fleas are readily obtained using growth on whole animal hosts, such as cats. Alternatively, fleas can be grown using artificial feeding systems such as those described by Wade, S.E., et al., J Med Entomol (1988) 25_:186-190. Preferred flea sources are those of the species Ctenocephalides felis, which is the most common species found infesting domestic animals. However, it is expected that cross-reactivity of antibodies prepared with respect to g_ felis will be found in other species. The membranes derived from either fed or unfed fleas may be used. Differences may be found in the characteristics of the preparation depending on the feeding status. By "fed" fleas is meant that the fleas from which the preparations are derived have been allowed to consume a blood meal for a 24-48 hour period either on animals or by artificial feeding prior to membrane preparation. In the case of "unfed" fleas, newly emerged fleas that have never taken a blood meal are used in the membrane preparations.

Total flea membrane (TFM) preparations are obtained by a number of alternative procedures. For example, frozen (about -70°C) unfed or fed male and/or fe ale fleas are disrupted by homogenization in buffer, e.g., 0.15M PBS, pH 7.2, 1 mM EDTA. A suitable proportion of fleas to buffer is about 2500 frozen fleas in 10 ml buffer. Homogenization can be conducted by shaking in the presence of 1/8" stainless steel beads at 4°C for 4 minutes, or by grinding the fleas to a powder with a mortar and pestle in the presence of liquid nitrogen prior to the addition of buffer. It may be helpful to sonicate the preparation, for example using a soniprep 150 (MSE) at an amplitude of 22 for a total of 3 1/2 minutes on ice (30 second bursts cooling on ice between bursts) .

Debris is then removed from the homogenate, usually by filtration through coarse filters or by spinning at low speed, for example 600 x g for 10 minutes at 4°C. The pellet may be rehomogenized and resonicated to obtain additional membrane extract.

The total supernatant is then freed of organ- elles and cells by centrifugation at moderate speed, for example at 15,000 x g for 20 minutes (at 4°C) to remove these nonmembranous portions, such as cells, mitochondria, lysosomes and microbodies. To harvest the membrane fraction, the supernatant from this spin (which contains the membrane fraction) is then centrifuged at very high speed, -100,000 x g for 2 hours at 4°C. The pellet from this harvest contains the membrane fragments including cell membranes and ribosomal and endoplasmic reticulum membrane fragments. The membrane pellet is then homogenized in a suitable buffer and assayed for protein content if desired. Individual antigenic components are isolated using conventional techniques for protein and glycoprotein separations such as ion exchange chromatography, gel filtration, affinity chromatography (e.g., using anti-flea sera as affinity ligand), gel electrophoresis and the like. To prepare membrane preparations from specific organs, prior to application of the aforementioned procedure, the fleas are dissected to obtain the desired tissue. For example, to obtain the midgut preparation, freshly hatched active unfed or fed male and/or female fleas are chilled on ice and placed on a drop of suitable buffer, such as the 0.15M PBS/1 mM EDTA buffer described above, on a glass slide. The midgut is removed by dissection under a binocular microscope, and immediately placed in a tube of the buffer on ice. The tubes are eventually stored at -70°C. It appears that -10,000 midguts are required to obtain a convenient preparation, although of course by modifying the procedure to a micro scale, smaller numbers could be used. The harvested midguts are thawed, pooled and homogenized in a glass/teflon homogenizer. The procedure described above for total membrane preparations may be applied, or alternatively the homogenate may be transferred to siliconized 1.5 ml icrofuge tubes and spun in an Eppendorf centrifuge at a setting 3 for 10 minutes at 4°C. The supernatants are transferred to other sili¬ conized tubes and spun at setting 13 for 15 minutes at 4°C. The supernatants are transferred to a Beckman 8.5 ml conical heat-seal tube overlaid with mineral oil and spun in an SW28 rotor at 100,000 x g for 2 hrs. The pellet of this spin is the membrane fraction and is assayed for protein content if desired, and the individual antigenic elements isolated if desired. In addition to midgut membrane, analogous preparations may be made from other internal organs, including ovary, testis, nerve cord and Malpighian tubules. Dissection according to the diagrammatic representation of internal flea anatomy in Figure 1 permits use of these various sources of internal organ membranes. For the experiments described herein, midgut preparations include the proventriculus (1) , the true midgut (2), the pylorus (6), and the hindgut (4). Also noted in Figure 1 are the locations of the Malpighian tubules (3) , the ovaries (5) , and the nerve cord (8) . In general, the preparation of suitable membrane-containing fractions involves homogenization of the organ desired or of the whole insect, removal of large components, and finally, removal of membranes from the soluble components by high speed centrifugation. Once harvested, the membrane fraction is useful as a source of purified antigen components which can be purified using immunoaffinity columns wherein the affinity ligand comprises antibodies raised by administration of the membranes, or other conventional separation techniques. Antigen-antibody complexes formed during such affinity-based purifications can also be used for immunization and protection.

For the antigenic components of the membrane extract which are of low molecular weight, conjugation to carrier to enhance immunogenicity is generally required. Suitable carriers include, for example, keyhole limpet hemocyanin (KLH) , animal or human serum albumin, and diphtheria or tetanus toxoid. Conjugation is by conven¬ tional means, depending on the nature of the antigen. The antigen can often be coupled directly to carrier, but more frequently a linker, such as those commercially available from Pierce Chemical Company, Rockford, IL, is used. The linkages employed are those conventionally used, such as amide bonds, thioethers, and disulfides. Alternatively, membrane antigens may be polymerized or crosslinked, for example, with gluteraldehyde, enhancing immunogenicity by increasing perceived molecular weight or presenting novel immunogenic epitopes.

In addition, mRNA prepared from the whole organism or the various tissues is available for reverse transcription into cDNA encoding said membrane antigens. Clones expressing this cDNA can be detected using the above antibodies; the cDNA can be ligated into expression vectors such as the lambda-gt system and the raised antibodies used to detect the production of antigen from these vectors. The induction of antibodies is described hereinbelow.

Induction of Antimembrane Antibodies and Vaccine Compositions

Depending on the nature of the membrane prepa¬ ration, the antigenic composition is administered in an immune protocol of multiple dosages at an appropriate protein concentration of 10-1000 μg per dose, preferably 100-250 μg, for total flea membrane (TFM) and at an appropriate dose, usually about 10-100 μg per dose for individual organ membranes such as midgut flea membrane (GFM) preparations. It is advantageous to use adjuvants, such as Quil A (saponin) , RIBI adjuvant, complete or incomplete Freund's adjuvant (CFA or IFA) , aluminum phosphate/alum, or one or more of the ISCOMs, or muramyl dipeptide (MDP) , depending on the subject. For administration of the vaccine to sheep, Quil A at l mg/ml in a total volume of 2.5 ml per injection is preferred. For use as a vaccine for cats, saponin at concentrations to 400 μg/ml or RIBI at suitable concentrations are used in a total volume of 0.5-1 ml per injection. Standard formulations for vaccines in general are usable and such formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton PA, latest edition. Administration is generally systemic by injection, usually intramuscular or subcutaneous injec¬ tion. Intravenous injection is, for the most part, impractical for veterinary use. Suitable subjects are those animals who are targets of flea parasitism. These are notably domestic animals such as cats and dogs, farm animals, such as cows, pigs, sheep and horses, and various domesticated birds, such as chickens. Experimental animals such as rabbits, rats and mice may also be immunized specifically for antibody production. The precise nature of the immunization regimen is readily determined using routine optimization procedures, and the general parameters for administration to a specific subject are well known to those of ordinary skill in the art. The dosage levels and the nature of the formulation, of course, varies with the nature of the subject and the severity of the infestation, as well as the precise vaccine active ingredient employed.

The results of vaccination as described above are severalfold. First, the vaccinated animals are protected against flea infestation in that the number of fleas found feeding on the animals directly subsequent to vaccination, even after challenge by artificially introducing a flea population, is greatly reduced. Also, feeding fleas that survive may have markedly reduced fecundity. Thus, the animals per se are protected from the parasitism of the insects. Second, the immunized animals are useful as sources of antibodies immunoreactive with the specific antigens in the membrane extracts. Antisera are recovered from these animals and used directly, for example, as affinity purification ligands or as detection reagents to analyze for the presence or absence_.of antigen, or the antibody-secreting cells such as spleen or peripheral blood lymphocytes are recovered, immortalized, and screened for their ability to secrete antibodies specific for the membrane antigens. Conventional methods to obtain monoclonal antibody-secreting cells using immunized animals are well known in the art. The immortalized cell lines can be screened using conventional immunoassays employing, as test antigen, the appropriate membrane extract or fraction or purified component thereof.

Third, by reducing the susceptibility of the vaccinated animal to feeding by the parasite, the viability of the flea population in the environment of the vaccinated animal is also reduced. Thus, not only is protection conferred on the animal per se, but the environment is de-infested by effective flea population control.

The following examples are intended to illus¬ trate but not to limit the invention.

Example 1 Production of TFM Vaccine A. Total flea membrane antigen (TFM) was prepared as described generally hereinabove. Briefly, about 5,000 frozen unfed C_ felis fleas were suspended in 20 ml of 0.15 M PBS, pH 7.2, containing 1 mM EDTA, and homogenized by shaking in the presence of 1/8-inch stainless steel beads at 4°C for 4 min. The beads were removed, and the mixture was then sonicated using a Soniprep 150 (MSE) at an amplitude of 22 for 3-1/2 min on ice using 30-sec bursts and cooling back to 6°C between bursts. The suspension was centrifuged at low speed (600 x g) at 4°C and the pellet was again treated as above to obtain additional extract. This extract was also recovered by centrifugation at low speed.

The combined supernatants were then centrifuged at moderate speed (about 15,000 x g) for 20 min at 4°C and the pellet was discarded. The supernatant was centrifuged at about 100,000 x g for 2 hr at 4°C and the pellet was recovered. The supernatant from this centrifugation step is designated TFS for total flea supernatant. The pellet is designated total flea membrane (TFM) . The TFM pellet was then homogenized in an additional 5 ml of 0.15 M PBS, pH 7.2, 1 mM EDTA. The final concentration of protein in the buffer was

0.1 mg/ml. A portion of the TFS and TFM preparations was subjected to gel electrophoresis on polyacrylamide gels using SDS detergent followed by Coomassie blue staining for protein visualization. The results of this gel are shown in Figure 2. Lane 1 shows total adult flea extract after the low speed spin. Lane 2 shows the TFM preparation. Lane 3 shows the TFS preparation.

B. About 3,000 unfed C^. felis fleas were ground with a mortar and pestle in liquid nitrogen, then homogenized in PBS 0.15M, pH 7.2 containing 1 mM EDTA and PMSF. Homogenization was in a Potter Elvehjem teflon homogenizer and was performed on ice. The homogenate was then centrifuged at 2000 x g for 10 minutes and the supernatant removed and centrifuged at 15,000 x g for 30 minutes. This supernatant was then centrifuged at 100,000 x g for 2 hours. All centrifugations were performed at 4°C.

The supernatant from this centrifugation was designated total flea supernatant (TFS) and put aside. In some experiments TFS was also used in vaccine preparations. The pellet was resuspended in PBS 0.15M, pH 7.2 with 1 mM EDTA and was designated total flea membrane (TFM) . This procedure yielded 35 ml of TFS containing 1-2 mg/ml protein, and 1 ml of TFM containing -2 mg/ml of protein. Portions of TFS and TFM were subjected to electrophoresis on polyacrylamide gels using SDS detergent followed by Coomassie blue staining for protein visualization (Figure 3). Lane 1 shows total adult flea extract after the low speed spin. Lanes 2 and 3 are TFS samples from two different preparations. Lanes

4 and 5 are TFM samples from two different preparations which demonstrate the reproducibility of the protein profile of TFM by this protocol.

C. To crosslink the TFM, and/or to conjugate it to carrier, the TFM suspended pellet was adjusted to exactly 2 mg/ml in PBS, and aliquoted into 2 x 100 μl. 400 μg keyhole limpet hemocyanin (KLH) powder was added to one aliquot, and 1 μl of 25% glutaraldehyde solution was added to both aliquots. The solutions were left for 2 hours at room temperature and diluted in PBS to 2 ml prior to inoculation of animals. The vaccine containing TFM treated with KLH and glutaraldehyde was designated TFM-KLH, that containing TFM treated only with glutaraldehyde was designated TFM-glut. Portions of these two preparations were also subjected to electrophoresis on polyacrylamide gels using SDS detergent, where it could be seen that both preparations had been extensively cross-linked, the majority of material now appearing as high molecular moieties at the top of the gel.

D. Vaccines were formulated with adjuvants recommended by manufacturer or known in the art.

Example 2 Protection of Sheep with TFM Vaccines Two groups of 4 sheep each were vaccinated with either 250 μg TFM, as prepared in Example 1A, in 2.5 ml vaccine containing l mg/ml Quil A, or with a control vaccine of 2.5 ml volume containing 1 mg/ml Quil A alone. The sheep were bled throughout the immunization regimen and antibody titer against flea membrane antigen was determined in a standard solid state ELISA, using the TFM preparation as antigen. After 6 immunizations the sheep were challenged as follows.

The back of the animal was shaved and a cup containing 30 fleas was affixed to the shaved area with glue. The fleas were allowed to feed for seven days after which time the contents of the cups were analyzed for the number of living and dead fleas. (In general, only 50% of fleas on sheep feed.)

The results obtained showed protection by the test vaccine of sheep from feeding by the fleas. In the controls, 29.3+0.5 fleas were recovered from the cup, of which 16+2.7 (or 55%) were dead and 13.3+3 were alive.

However, in the TFM-vaccinated sheep, 28+0.4 fleas were recovered per cup of which 18.9+3.3 (or 67.5%) were dead; only 8.9+3.3 remained alive. Thus, protection due to the presence of TFM in the inoculum was shown.

Example 3 Efficacy of TFM Vaccine in Cats Conditioned adult cats of mixed breed and sex were subjected to a "prechallenge" with 100 unrestrained fleas in order to determine the number of fleas each individual cat would carry through a seven-day challenge period prior to any treatment such as immunization. Two groups of four cats were vaccinated with TFM, prepared as in Example IB, using 200 μg protein per 0.5 ml dose and either saponin or RIBI adjuvants for the primary injection, followed by 100 μg of protein for all subsequent immunizations. Four adjuvant control cats all received saponin alone.

The animals were again challenged with 100 unrestrained fleas after the fourth immunization. For one week subsequent to challenge, cat pans and cats were visually checked for fleas daily. At seven days post- challenge, living fleas were combed off and enumerated. and individual cat ova-cultures were set up from the contents of each pan.

Following challenge, the four RIBI adjuvant- TFM cats were split into 2 groups of two and immunized with either TFM-KLH or TFM-glut (Example 1C) . A second immunization was performed two weeks later and the cats were challenged three weeks later and then again at four weeks. The results of the challenge experiments are shown in Table 1.

Table 1

% FLEAS RECOVERED CHALLENGE

CAT # IMMUNIZED WITH £2 #1

1479 TFM x 4, 15 16 TFM-KLH X 2, All RIBI

1372 TFM X 4, 34 32 TFM-KLH X 2 , All RIBI

1478 TFM X 4, 11 TFM-Glut X 2, All RIBI

1509 TFM X 4, 11 TFM-Glut x 2, All RIBI

Control Unimmunized 37 29

The results obtained showed protection by TFM followed by TFM-glut on two separate challenge occasions, Flea recovery is expressed as the percentage of fleas recovered after challenge with 100 unrestrained fleas. On challenge number 2, 37% of fleas were recovered from the control cat, whereas only 4% and 8% were recovered from the TFM-glut cats. On challenge number 3, 29% of fleas were recovered from the control animal, while 11% were recovered from each of the TFM-glut cats. In addition, one TFM-KLH animal (1474) was protected in both challenge 2 and challenge 3, 15% and 16% of fleas being recovered respectively. It is important to note that the differences between these animals do not correspond to preening differences between animals as seen in the pre- immunization challenge.

Example 4

Production of GFM Cat Vaccine A. All procedures are conducted on ice. Using a "glass homogenizer" tissue or Dounce, 1000-1200 unfed flea midguts were homogenized on ice using several strokes until suspension was opaque and no pieces were visible. The suspension was then sonicated with a high energy unit once for 20 sec. Temperature measured in the sonicating buffer alone reaches 6-10°C during the sonication. The sonicated suspension was spun § 15,000 x g for 20 min at 4°C, and the supernatant (SI) was retained. The pellet (-1200 midguts) was resuspended in 2 ml antigen buffer by Vortex, and the glass homogenizer was again used to obtain an opaque suspension. The suspension was sonicated 7 times for 20 sec with 2 min rests for cooling. The sonicate was spun at 15,000 x g for 20 min at 4°C to obtain a second supernatant (S2) . In some cases, the pellets from the 15,000 x g spins were reextracted for additional protein. Otherwise they were discarded. The two supernatants SI and S2 above were combined and centrifuged at 100,000 x g for 2 hr. The resulting supernatant is designated gut flea supernatant (GFS) and has a volume of 96 ml at 82 μg protein/ml, i.e. 7.9 mg total protein.

The pellet from the 100,000 x g spin is designated gut flea membrane (GFM) and is resuspended by glass homogenizer in 2 ml buffer. From 11,000 midguts, this pellet (diluted to 6 ml) gave 928 μg protein of which, after withdrawal of aliquots for assay, 836 μg remained.

B. In another method designed to obtain higher protein yield, GFM was prepared with a single centrifugation step. Two hundred tubes of 100 dissected unfed midguts each were thawed on ice and centrifuged in an Eppendorf microfuge for 5 minutes at maximum speed at 4°C. Approximately 100 μl of supernatant was removed from each tube, pooled, and designated "low speed supernatant". Each pellet was resuspended in 50 μl of flea antigen buffer (1 x PBS, 1 mM EDTA) . In groups of 1000 organs, the suspended pellets were transferred to a 1 ml conical glass/glass homogenizer and homogenized on ice to a smooth suspension (-17 strokes) . In aliquots of 2000 organs, each prep was sonicated in an ice water bath for 2 minutes in 15 second bursts at 40% power using a microprobe. The sonicated material was centrifuged in 2 aliquots (10,000 organs each) in a Beckman type 65 rotor for 2 hours at -100,000 x g (4°C) . The "high speed" supernate from the 100,000 x g spin was added to the low speed supernatant for a total volume of 35 ml. This was designated gut flea supernatant (GFS) . Total protein in GFS from 20,000 organs was 2.88 mg.

The two membrane pellets were resuspended in 200 μl flea antigen buffer each and frozen. Pellets were thawed, resuspended in a total volume of 2 ml, sonicated at 40% power for 2 minutes in 15 second bursts in ice water bath, and diluted to a final volume of 5 ml. This preparation was designated GFM. The total GFM protein yield from 20,000 organs was 2.5 mg. Figure 4 shows a silver-stained polyacrylamide gel of GFS (lane 1) and GFM (lane 2) .

Example 5 Efficacy of GFM in Cats

A. Ten flea-free cats of mixed breed and sex were group-housed in a clean environment and "prechallenged" twice with 50 fleas each in order to determine their natural preening activity. The ten cats were randomized in two test groups in order to account for individual variance in preening activity. The two test groups were:

5 cats receiving GFM immunization;

5 cats receiving adjuvant control immunization. Antigen preparations were as follows. The first immmunization (week 0) contained 10 μg GFM as described in example 4A with 500 μg Quil A adjuvant in 2 mis total volume. The second immunization (week 4) contained 10 μg GFM with 1 mg Quil A adjuvant in 2 ml total volume. The third immunization (week 8) contained 25 μg GFM with RIBI adjuvant (prepared per manufacturer) in 1 ml total volume. Four subsequent boosts contained varying amounts of GFM with RIBI adjuvant in 1 ml volumes. For adjuvant control immunizations, cats received adjuvant concentrations as described for each

GFM immunization, but without antigen. Animals were bled throughout the study and challenged when cat serum ELISA titers against TFM plateaued.

The cats were challenged with flea infestation as described in Example 3. The results were evaluated in terms of the percentage of fleas recovered after vaccination in comparison with the percentage of fleas recovered prior to vaccination in vaccinates and the control groups. The percentage of fleas combed off after vaccination were, on the average, 32% lower (p<0.1) than the percentage recovered before, whereas in the control groups the fleas combed off at a similar time were only 7% lower than the previous percentage. In comparing the flea counts of vaccinated versus unvaccinated cats at comparable "postvaccination" times, the vaccinates harbored, on the average, 33% fewer fleas.

B. Twenty flea-free weanling cats of mixed breed and sex obtained at age 6 weeks were housed two per cage in a clean environment until they reached twelve weeks of age. Sixteen cats were selected based on weight and sex, pre-bled, tattooed, and placed two per cage in groups of:

6 cats receiving GFM immunization; 6 cats receiving GFS immunization;

4 cats receiving adjuvant control immunization. Antigen preparations were combined with RIBI R-730 adjuvant as follows:

For GFM, 0.33 ml of the 5 ml total GFM preparation described in Example 4B with 1.7 ml of flea antigen buffer (1 x PBS with EDTA) was combined in one vial (x3) of RIBI and vortexed for three min. One ml (approximately 80 μg protein) was administered intramuscularly to each of 6 cats. For GFS, 2 ml of the total 35 ml combined super tant preparation of Example 4B was added to one vial (x3) of RIBI and vortexed for 3 min. One ml (approximately 80 μg protein) was administered intramuscularly to each of 6 cats.

For adjuvant control, 2 ml of flea antigen buffer was added to 1 vial of RIBI (x2) and vortexed for 3 min. One ml was administered intramuscularly to each of four cats.

All cats were immunized as above on day 0 and at 21-day intervals thereafter up to 5 immunizations. Cats were bled throughout the study. Titers to TFM in each cat's sera are measured by ELISA. One to two weeks after the last antigen boost, each cat is challenged as in Example 3, by being infested with 100 unfed fleas. After 1 week, fleas are combed off each cat and counted. Cat pans are set up and ova-cultures done from each pan's contents. All cats are bled out and sera from those experimental animals which demonstrated protection is used as a molecular screening reagent.

c. Following the procedure in paragraph A of this example, cats are immunized with GFM prepared as described, except using fed rather than unfed fleas as starting material and the results noted.

Claims

l. A composition for protection of a mammalian or avian subject against infestation by fleas, which vaccine comprises an amount of fed or unfed flea membrane or fed or unfed flea membrane extract or effective antigenic component thereof, effective to confer resistance to said infestation.

2. The composition of claim 1 wherein said membrane is total membrane or is derived from flea midgut, ovary, testis, nerve cord or Malpighian tubule.

3. Antibodies specifically immunoreactive with the composition of claim 1.

4. An antigen-antibody complex consisting essentially of the antigenic component of the composition of claim 1 and an antibody specifically immunoreactive therewith.

5. A method to reduce the flea population in the environment of an avian or mammalian subject, which method comprises administering to said subject an effective amount of the composition of claim 1.

6. A method to prepare a composition effective in protecting mammalian or avian subjects against flea infestation which method comprises preparing a homogenate of fed or unfed flea tissue; separating the homogenate into a fraction containing debris and organelles and a fraction that is a first supernatant; recovering the first supernatant and separating the supernatant into a fraction containing membranes and a second supernatant; and recovering the membranes and fragments and extracting the antigenic components from said membranes and fragments.

7. The method of claim 6 which further includes affinity chromatography which comprises applying the antigenic-component-containing extract to an affinity support wherein the affinity ligand is an antibody or fragment thereof specifically immunoreactive with an antigenic component so as to adsorb said antigenic component by forming a complex with said antigenic component followed by eluting said complex or said antigenic component from the support.

8. A complex of an antibody or fragment thereof with an antigenic component of flea membrane or said antigenic component prepared by the method of claim 7.

9. The method of claim 6 wherein the membranes and fragments are recovered by centrifugation of said supernatant at -100,000 x g to obtain a second super¬ natant and a pellet, and recovering the pellet.