WO1999002182A2

WO1999002182A2 - Non-aqueous vaccines

Info

Publication number: WO1999002182A2
Application number: PCT/IB1998/001327
Authority: WO
Inventors: Mark Albrecht; Richard William Brown; James Hungerford
Original assignee: Biotech Australia Pty. Limited
Priority date: 1997-07-10
Filing date: 1998-07-10
Publication date: 1999-01-21
Also published as: WO1999002182A3; AR013202A1; UY25089A1

Abstract

A single-phase, non-aqueous vaccine formulation contains an immunogenic polypeptide in a polar, non-aqueous solvent. The formulation also may contain adjuvants and/or pharmaceuticals which may be coadministered with the immunogenic polypeptide. Methods of preparing such a vaccine formulation are disclosed, and methods of using the formulations to prevent and treat disease in mammals are provided.

Description

NON-AQUEOUS VACCINES

BACKGROUND OF THE INVENTION

Traditional, liquid vaccines contain an antigen in an aqueous carrier solution that also contains an adjuvant. The adjuvant typically may be a water soluble component (such as Quil A), an oil emulsion (such as an oil in water emulsion) or a mineral salt (such as aluminum hydroxide). Traditional solid vaccines contain an antigen entrapped in a solid adjuvant matrix (such as microspheres or pellets) that are suspended or dissolved in aqueous carriers prior to use.

Typically, conventional vaccine formulations contain more than 20%, and more usually more than 45%, v/v water. Formulations containing lower amounts of water invariably have been considered unsuitable for use as vaccines because the antigen components in the vaccine are thought to be unstable.

Available aqueous formulations suffer from numerous drawbacks, however_. For example, long term storage of the formulation typically requires special measures, such as refrigeration, to maintain stability of the formulation. This is especially disadvantageous, for example, in developing countries where adequate refrigeration for vaccine storage can be problematic. Similarly, in veterinary applications for vaccination of livestock, a requirement for refrigeration vaccine is highly inconvenient, and adds to the cost of livestock vaccination.

Alternatively, some vaccine formulations may be stored in a dried state, which requires an additional step of dilution with sterile aqueous solvent prior to administration. This essentially doubles the effort required to administer the vaccine, which is highly inconvenient, for example, in vaccination programs in developing countries where, where large numbers of people must be vaccinated. Similarly, for livestock vaccination, each additional step in the vaccination process reduces the economic efficiency of the vaccination process.

In addition, it would be highly advantageous to be able to co-administer a vaccine with a pharmaceutical. This would significantly reduce the workload, for example, in treating livestock, and could lead to significant economic advantages. For example, in developing countries it would be highly advantageous to be able to administer "single-shot" treatments that would combine one or more vaccines with one or more drugs. The drug and the vaccine could be targeted against the same disease or against different diseases. For example, a vaccine against tuberculosis or malaria might be co-administered with an anthelmintic or anti- schistosomal drug.

Similarly, in many parts of the world it is common to vaccinate livestock against infestation with parasites such as ticks and nematodes. Following vaccination, a significant time lag occurs before a significant reduction in parasite load is observed, due to the time taken for induction of an immune response against the parasite. Because of this time lag, farmers often use an additional antiparasitic drug to reduce parasite infestation during the time lag. But many pharmaceuticals are partially or completely insoluble, or are unstable in, aqueous solution. Accordingly, the pharmaceuticals cannot be admixed with an aqueous vaccine for a "single shot" administration, and separate administration of the vaccine and antiparasitic drug is required, leading to increased work and higher cost.

It is apparent, therefore, that new liquid vaccine formulations having enhanced stability and greater ease of administration are greatly to be desired. It also is apparent that formulations that can be used to simultaneously administer a vaccine and one or more pharmaceutical compounds are highly desirable.

SUMMARY OF THE INVENTION

It therefore is an object of this invention to provide single-phase non- aqueous vaccine compositions that may be stored without refrigeration.

It is a further object of this invention to provide a single-phase non- aqueous pharmaceutical formulation comprising an immunogenic polypeptide and a pharmaceutical in a non-aqueous solvent.

It is yet another object of the invention to provide a single-phase non- aqueous vaccine formulation prepared by admixing an immunogenic polypeptide with a polar non-aqueous solvent. It is still another object of the invention to provide methods of manufacturing these vaccine formulations.

It is a further object of the invention to provide methods of treating mammals by administering the vaccine formulations. In accomplishing the foregoing objects, there has been provided, in accordance with one aspect of the present invention, single-phase non-aqueous vaccine compositions comprising an antigenic polypeptide in a polar non-aqueous solvent. These formulations may be stored at ambient temperature while retaining efficacy. In a preferred embodiment, the non-aqueous solvent comprises dimethyl sulfoxide.. In another preferred embodiment, the non-aqueous solvent further comprises a non-aqueous hydroxylic solvent. In one embodiment, the hydroxylic solvent is selected from the group consisting of glycerol formal and propylene glycol, and in another embodiment, the hydroxylic solvent comprises a mixture of glycerol formal and propylene glycol. In further embodiments, the dimethyl sulfoxide comprises up to about 6% v/v, 3-6% v/v, or preferably about 3% v/v of the non-aqueous solvent. In yet another embodiment, the volume ratio of glycerol formal to propylene glycol is about 40:60.

In yet another embodiment, the formulation further comprises at least one non-aqueous adjuvant. In one embodiment, the adjuvant is selected from the group consisting of saponins, mineral or non-mineral oils, detergents, and pluronic polymers. In a preferred embodiment, the adjuvant is Marcol 52, and comprises up to about 12.5% v/v of the non-aqueous solvent. In another embodiment, the adjuvant is Pluronic Polymer, and comprises up to about 5% v/v of said non-aqueous solvent. In another embodiment, the adjuvant comprises a mixture of Marcol 52, containing up to about 12.5% v/v of the non-aqueous solvent, and Pluronic Polymer, containing up to about 5% v/v of the non-aqueous solvent.

In accordance with another aspect of the invention, there has been provided a single-phase non-aqueous pharmaceutical formulation comprising an immunogenic polypeptide and a pharmaceutical in a non-aqueous solvent. In one embodiment, the pharmaceutical is an endectocide, and in a preferred embodiment, the endectocide is an anthelmintic. In another preferred embodiment the anthelmintic is a macrocyclic lactone of the milbemycin or avermectin group of anthelmintics. In yet another preferred embodiment, the anthelmintic is abamectin or ivermectin

In accordance with a further aspect of the invention, there have been provided methods of manufacturing the vaccine formulations, comprising admixing an immunogenic polypeptide with a polar, non-aqueous solvent, or by admixing an immunogenic polypeptide and a pharmaceutical with a polar, non- aqueous solvent

In accordance with another aspect of the invention, there has been provided single-phase non-aqueous vaccine formulations prepared by admixing an immunogenic polypeptide with a polar non-aqueous solvent, or by admixing an immunogenic polypeptide and a pharmaceutical with a polar non-aqueous solvent In accordance with another aspect of the invention, there has been provided a method of vaccinating a mammal, comprising administering to the mammal an effective dose of the vaccine described above In one embodiment of the vaccine formulations, the immunogenic polypeptide is derived from a parasitic organism, and preferably is derived from an organism selected from the group consisting of, nematodes and insects In another embodiment, the polypeptide is derived from a tick selected from the group consisting of Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Amblyomma spp, Dermacentor spp, Ixodes spp, and Hyakmma spp In a preferred embodiment the tick is selected from the group consisting of Boophilus microplus, Boophilus annulatus, Boophilus decoloratus, Otobius megnim, Rhiphicephalus appendiculatus, Dermacentor andersoni, Dermacentor variabihs, Haemaphysalis longcorms, Amblyomma variegatum and Ixodes holocydus) In a further embodiment, the immunogenic polypeptide is Bm86

In yet another embodiment, the immunogenic polypeptide is derived from a haematophageous parasitic insect selected from the group consisting of Haemotobia spp, Hypoderma spp, Dermatobia spp, Anopheles spp, Luciha spp, Ctenocephahdes spp, Chrysomya spp, Gasterophulus spp, Cuhcoides spp, Stomoxys spp, Lmognathus spp, Solenoptes spp, Haematopinus spp, Melophagus spp, Aedes spp, and Culez spp In another embodiment, the immungenic polypeptide is derived from a nematode selected from the group consisting of Strongylus spp, Tr chostrongylus spp, Haemonchus spp, Ostertagia spp, Ascaris spp, Trichinella spp, Toxascaris spp, Uncinaria spp, Ancylostoma spp, Trichuris spp, Dirofilaria spp, Necator spp, Ancylostoma spp, Ascaris spp, Trichuris spp, Enterobius spp, Strongyloides spp, and Wuchereria spp. In a preferred embodiment, the nematode is selected from the group consisting of Strongylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostartagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonna, Uncinaria stenocephala, Ancylostoma caninum, Trichuris vulpis, Dirofilaria immitis, Necator americanus, Ancylostoma duodenale, Asceris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals and Wuchereria bancrofti.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides single-phase, non-aqueous vaccine compositions that are useful for prophylaxis and for treating disease in mammals. The compositions contain an immunogenic polypeptide antigen in a polar non- aqueous solvent. In addition non-aqueous vaccines are provided that further contain a pharmaceutical compound. The invention also provides methods of preparing the non-aqueous vaccines, and methods for their use in treating mammals. Surprisingly, it has been found that immunogenic polypeptides can be administered to mammals in polar non-aqueous solvents without significantly decreasing the efficacy of the observed immune response against the polypeptide. Moreover, the use of polar non-aqueous solvents allows the preparation of vaccine formulations that also contain pharmaceutical compounds that are soluble in the solvents, which therefore can be coadministered with the vaccine. The formulations are observed to have enhanced stability in the absence of refrigeration compared to traditional aqueous vaccines. In particular, the formulations can be stored for prolonged periods at ambient temperatures of up to about 40°C while retaining efficacy, i.e. the ability to induce a protective immune response. The formulations also are more convenient to use than dry vaccines that must be diluted in a separate step prior to administration. The invention is particularly useful for applications in developing countries, where large-scale vaccination programs require efficient methods of vaccinating large numbers of people as quickly and inexpensively as possible. The invention also is useful for veterinary applications.

In a preferred embodiment, the non-aqueous solvent comprises dimethyl sulfoxide (DMSO), although the skilled artisan will recognize that other polar non-aqueous solvents also can be used DMSO is non-toxic, and has been used in humans and other animals for treatment of a variety of ailments. Sterile, purified DMSO suitable for use in the present invention is commercially available from, for example, Sigma Corp. (St. Louis, MO), although any pharmaceutical grade DMSO may be used. The skilled artisan will recognize that methods of purifying and sterilizing DMSO are well known in the art.

Advantageously, the DMSO comprises up to about 6% of the non-aqueous solvent, with the remained comprising a polar hydroxylic solvent. Monohydroxylic solvents suitable for use in the invention include glycerol formal, (GF, which typically comprises a mixture of 5-hydroxy-l,3,-dioxane and 4- hydroxy methyl- 1,3, dioxolane) and benzyl alcohol. Polyhydroxylic solvents suitable for use in the invention include propylene glycol. (PG), and other solvents that are miscible with DMSO. Advantageously, a mixture of PG and GF is used. For example, the proportion of PG:GF can vary between about 10:90 to about 90: 10 (v/v), although ratios outside this range also can be used. Most advantageously, the ration varies between about 60:40 and 40:60. Methods of varying the ratios of PG and GF to achieve optimal solubility and immunogenic efficacy (immunogenicity) of the vaccine components are routine and are within the purview of the skilled artisan. For example, solubility can be assayed by preparing a solution of the polypeptide in a certain ratio of a hydroxylic solvent mixture, and subjecting the solution to high speed centrifugation. The quantity of precipitate pellet observed following centrifugation provides a measure of the solubility of the polypeptide in that ratio of hydroxylic solvents. The immunogenic efficacy of the composition can be determined simply by measuring the antibody titre generated by the various compositions, with a higher titer being indicative of higher immunogenicity. In a preferred embodiment, the non- aqueous solvent comprises about 3% DMSO in about 60:40 PG/GF.

The non-aqueous vaccine also may contain an adjuvant to enhance the immune response against the immunogenic polypeptide. Any non-aqueous adjuvant that is approved for use in humans or other animals may be used in the invention, provided it is miscible with or can be emulsified with the non-aqueous solvent. Such adjuvants are well known in the art and include saponins such as Quil A (Superfos, Vedbach, Denmark), mineral oils such as Marcol 52 (Esso, Sydney, Australia), non-mineral oils, such as Montanide 103 (SEPPIC, Paris, France), and pluronic polymers, such as L121 (BASF, NJ). Other adjuvants that may be used include squalane or squalene, Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate), surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N,N'-bis(2-hydroxyethyl)-propanediamine, methoxy- hexadecylglycerol and pluronic polyols, polyanions such as pyran, dextran sulfate, polyacrylic acid and carbopol, peptides and amino acids such as muramyl dipeptide, dimethylglycine, tuftsin and trehalose dimycolate.

The amount of adjuvant that may be used in the compositions will vary depending on the nature of the adjuvant. The skilled artisan readily is aware of the amounts of known antigens that must be used to obtain a desired immune response, and optimization of the amount of adjuvant is routine. For example, a typical proportion of Marcol 52 in the non-aqueous vaccine formulation solvent of the invention is about 12.5% v/v. In another example, the proportion of Pluronic Polymer is about 5%. Combinations of adjuvants also may be used, and the relative proportions of each adjuvant adjusted accordingly. Optimization of the relative proportions of adjuvant is routine for the skilled artisan. Considerations in determining optimum adjuvant proportions include immunogenicity of the polypeptide, extent and duration of side effects of vaccination (for example, size of local reactions at the injection site), and effect on the solubility of the polypeptide.

As applied herein with respect to vaccine compositions, the term "non- aqueous" indicates that the compositions are free of exogenous or added water, other than residual trace water that is present in either the immunogenic polypeptide or the DMSO. It is well known that polypeptide compositions that have been dried by lyophilization or by other methods contain trace or residual water that is difficult or impossible to remove. Similarly, DMSO has a high affinity for water, and commercial preparations of the solvent may contain small but measurable amounts of residual water. In the context of the invention, the presence of trace or residual water in either the immunogenic polypeptide or the DMSO is expected; however, vaccines containing these trace amounts of water are to be considered "non-aqueous for the purpose of the invention.

In the context of this description, qualifying a vaccine formulation as

"single phase" means that the immunogen of the formulation is sufficiently soluble in the non-aqueous solvent that it cannot be separated from the solvent by mechanical means. For example, high-speed centrifugation of a single-phase vaccine does not produce therefrom a significant pellet of an immunogenic polypeptide.

The immunogenic polypeptide used in the present invention can be any polypeptide that is capable of generating a protective immune response in the host mammal. The skilled artisan will appreciate that a large number of such immunogenic polypeptides are known in the art The term "polypeptide" is employed here to denote any peptide having two or more amino acids linked by peptide bonds. The immunogenic polypeptide may contain a single polypeptide chain, or may contain multiple polypeptide chains linked together, for example, by disulfide bonds or by other chemical cross-links. Polypeptides chains containing less than about 50 amino acids produce only a weak immune response due to their small size. Typically, these small polypeptides are linked to a larger carrier protein, such as serum albumin, to enhance the immune response against the immunogenic polypeptide. Methods of linking polypeptides to carrier proteins are well known in the art. Preferred single chain polypeptides for the present invention typically contain over 75 amino acids and, more typically, over 150 amino acids.

Advantageously, the immunogenic polypeptide is derived from an organism that causes disease or other adverse health effects in humans or other mammals. The disease-causing organism may be a bacterium, virus, or other microbe, or may be a parasitic organism, such as a tick or other insect, or a parasitic worm, such as nematodes. In particular, it is desirable to use immunogenic polypeptides derived from ticks such as species of Boophilus, Haemaphysalis, Otobius, Rhiphicephalus, Amblyomma, Dermacentor, Ixodes, and Hyakmm. More particularly, the polypeptide may be derived from Boophilus microplus, Boophilus annulatus, Boophilus decoloratus, Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, Dermacentor variabilis, Haemaphysalis longcornis, Amblyomma variegatum ox Ixodes holocydus). The polypeptide also may be derived from parasitic nematodes such as species of Strongylus, Trichostrongylus, Haemonchus, Ostertagia, Ascaris, Trichinella, Toxascaris, Uncinaria, Ancylostoma, Trichuris, Dirofilaria, Necator, Ancylostoma, Ascaris, Trichuris, Enterobius, Strongyloides, and Wuchereria. In particular, the polypeptide may be derived from Strongylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostartagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonna, Uncinaria stenocephala, Ancylostoma caninum, Trichuris vulpis, Dirofilaria immitis, Necator americanus, Ancylostoma duodenale, Asceris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wuchereria bancroftϊ). The polypeptide may also be derived from haematophageous parasitic insects, for example, species of Haemotobia, Hypoderma, Dermatobia, Anopheles, Lucilia, Ctenocephalides, Chrysomya, Gasterophulus, Culicoides, Stomoxys, Linognathus, Solenoptes, Haematopinus, Melophagus, Aedes, or Culez In a particularly preferred embodiment, the immunogenic polypeptide is derived from Boophilus microplus. A so-called "concealed antigen" derived from B. microplus, denoted "Bm86," is particularly suitable for use in the invention. Bm86 has been shown to be highly protective against parasitic infestation when administered to animals. A commercial formulation of Bm86, known as "TickGard Plus" is marketed by Biotech Australia Ltd. under the trademark "TickGARD." The gene encoding Bm86 was sequenced, and a method for large-scale bacterial expression also has been described. See Willadsen et al, Immunology 143: 1346 (1989); Rand et al, Proc. Nat'lAcad. Sci. USA. 86:9657 (1989), and U.S. patent No. 5,587,311. In addition to bacteria, Bm86 also may be expressed in high yield in yeast.

The term "derived" is employed here, in relation to the immunogenic polypeptides of the present invention, to denote those obtained by isolation and purification from a disease-causing organism., such as a tick, insect or nematode life stage which expresses the polypeptide, as well as antigens obtained by manipulation and expression of nucleotide sequences prepared from the organism. "Derived" also encompasses polypeptides produced from nucleotide sequences that encode the polypeptide, including genomic DNA, mRNA, cDNA synthesized from mRNA, and synthetic oligonucleotides. It further encompasses synthetic polypeptides prepared on the basis of the known amino acid sequences of the immunogenic polypeptides produced by the disease-causing organism. The polypeptide may have the same amino acid sequence as all or part of a protein expressed in the disease-causing organism, or the amino acid sequence may be modified to enhance the immune response against the organism. Methods of modifying amino acid sequences to enhance an immunogenic response are well known in the art. The polypeptide may or may not be glycosylated , and/or may contain other post-translational modifications.

Routes of administration of the non-aqueous vaccine formulations, dosages to be administered, and frequency of injections are factors that can be optimized using ordinary skill in the art. Preferably, the vaccine is administered parenterally, more preferably by subcutaneous injection. Typically, an initial vaccination is followed some weeks later by one or more "booster" vaccinations, the net effect of which is the production of vigorous cellular and humoral immune response against the immunogenic polypeptide. The present invention also contemplates non-aqueous vaccine formulations that contain a pharmaceutical compound. The pharmaceutical compound may be any compound that is soluble in the vaccine formulation, and that is suitable for co- administration with a vaccine, for example, by subcutaneous injection. The invention is particularly useful for coadministration of pharmaceuticals that are unstable in aqueous solution, although the invention is not limited to such compounds. Use of non-aqueous formulations prevents degradation of the pharmaceutical by hydrolysis and significantly improves the stability of the pharmaceutical. The formulations may be stored for prolonged periods at ambient temperatures of up to about 40°C while retaining the activity of the pharmaceutical Advantageously, the pharmaceutical compound is an anthelmintic compound, and more advantageously is a macrocyclic lactone of the avermectin or milbemycin group of compounds. In a preferred embodiment, the compound is Abamec™, which is a mixture of avermectins B 1 a and Bib. The skilled artisan will recognize, however, that the invention is not limited to avermectins or milbemycins Other suitable avermectins and milbemycins are described in EP 0750907A2, EP 0413538A1, WO96/37178, EP 0525307, and U.S. patents No. 4,853,372 and No. 4,389,397.

The pharmaceutical compound may be present in the non-aqueous vaccine formulation in any proportion that maintains the solubility of both the immunogenic polypeptide and of the pharmaceutical. When the pharmaceutical is an avermectin or milbemycin compound, it typically is present at a concentration of between 1- lOOmg /ml, although concentrations outside this range also may be used. Typically, the avermectin or milbemycin compound is present at a concentration of about lOmg/ml.

The present invention, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration as a preferred embodiment but is not intended to be limiting of the present

EXAMPLES

Vaccine formulations containing antigen and adjuvant were compared to the commercial vaccine TickGARD Plus for efficacy in generating antibodies against recombinant protein antigen Bm86. Further comparison was made between plasma abamectin level generated by the novel formulations and that generated by injection of Avomec, a conventional abamectin formulation.

1. General Methods

TickGARD Plus is a commercially available vaccine containing Bm86 antigen at a concentration of 25μg/ml. Avomec was administered at a dose of 2ml/100kg, containing lOmg abamectin/ml (to give 200μg abamectin/kg).

TickGARD plus and Avomec were administered to the same animals on opposite sides of the neck, allowing a reduction in the number of control animals required.

Abamectin was from Haimen Pharmaceutical Factory, China, an approved source of abamectin for use in veterinary products. Abamectin supplied by Haimen is 95% pure, with a ratio of avermectin Bla to B lb of 96:4.

a. Animals

Cattle were allocated to each treatment regimen such that the average body weight were similar for each group. The animal trial work was approved by the Animal Care and Ethics Committee of Biotech Australia Pty Limited. The animals were kept in paddocks with improved pasture and were also provided with supplementary feed (hay and cereal-based concentrate). The animals were handled in yards equipped with a race and single-animal crush. The cattle were weighed using load cells ("Rudweigh") attached to the crush. Various beef breeds were used in the efficacy trial. The age varied from 1 to 2 years of age, and start weight varied from 230kg to 450kg. The cattle were identified by ear tag and had been on a commercial farm for at least 2 weeks prior to the commencement of the trial. The cattle had not been treated with an endectocide within the last 6 weeks to 4 months prior to starting the trial.

b. Administration of vaccines For animals receiving Avomec or TickGARD Plus, 2ml/100kg Avomec was delivered subcutaneously to the right side of the neck, while 2ml TickGARD Plus was delivered to the left. For those groups receiving the combination formulations, 1 or 2ml/100kg of the appropriate formulation was administered by subcutaneous injection to the left side of the neck. A single injection was delivered on days 1 and 29. The size of the injection site reactions on the cattle in the trial was assessed for the initial 15 days after treatment, and inflammation or drainage at the site was noted. Any other adverse reactions (changes in appetite, demeanor, etc) were noted whenever the animals were handled for blood sampling.

c. Blood sampling

For blood sampling, 20 - 70ml of blood was collected from the jugular or the tail veins using a 18 gauge needle and vacutainer tube. Blood was centrifuged within 1 hour of collection, then the plasma divided into 3 ml aliquots and frozen to - 20°C within 4 hours of collection. The plasma was kept frozen until assayed. Antibody titer was determined using an ELISA assay that specifically recognizes B m86.

d. Abamectin assay

Serum abamectin concentrations were determined using an HPLC method with fluorescence detection. After addition of an internal standard (ivermectin), serum samples were prepared for analysis by protein precipitation followed by solid phase extraction. Conversion to the fluorescent derivative was accomplished by treatment with a trifluoroacetic anhydride solution. The fluorescent derivatives were separated and quantitated by narrow bore reversed phase HPLC (C 18). This method has a sensitivity of approximately lmg abamectin/2ml plasma and it quantifies total abamectin (avermectin B la + avermectin Bib).

e. Abamectin stability Aliquots of each formulation were stored at either 4°C or 40°C for periods up to 8 weeks to assess the stability of the abamectin. The level of abamectin in each formulation then was quantified in-house by HPLC analysis with UV detection (which quantifies abamectin B la and Bib separately).

f. Statistical analysis

Titers were logio transformed prior to analysis to normalize their distribution. The area under the abamectin serum curve (from day 1 to day 22) was estimated using the trapezoid rule.

Abamectin levels were assessed as two separate parameters: (i) by recording the maximum abamectin level for each animal, and (ii) by comparison of the abamectin level integrated over days 1 through 22 using Kruskal-Wallis non-parametric one way analysis of variance. Inclusion of weight as a covariant did not significantly reduce the residual error. Accordingly, weight was excluded as a covariant in the studies. Antibody titers were assessed by comparing maximum group geometric mean titers by ANOVA.

EXAMPLE 1: Comparison of aqueous and non-aqueous vaccine formulations. This trial was aimed at demonstrating the ability of the novel formulations to elicit antibody production similar to TickGARD Plus against the tick antigen, Bm86. The novel formulations contained Bm86 and adjuvant and were used as boosters for previously vaccinated cattle. Bm86 antibody titer generated by two novel formulations were compared to the control product TickGARD Plus. Table 1. Formulations

Formulation and Dosage Composition (per lOOmL. volumes are rounded)

TickGARD Plus Standard commercial TickGARD Plus batch TG03E007 Dosage: 2ml animal

Organic - Novel Formulation DMSO: 3.0ml (contains 2.5mg Bm86, 50mg Dosage: lml/lOOkg adjuvant) GF: make up to lOOml glycerol formal

OrgVwater - Novel Formulation water: 10ml (contains 2.5mg Bm86, 50mg Dosage: lml/lOOkg adjuvant) GF: make up to 100ml glycerol formal

Table 2. Group geometric mean Bm86 antibody titers

Weeks Past Boost

Vaccine 0 2 4

TickGARD Plus 2724 10,160 7474

Organic - Novel Formulation 1532 7130 5371

Org/water - Novel Formulation 2890 6579 2921

No significant difference was observed between the levels of anti-Bm86 titers generated by the commercial vaccines and those generated by the non-aqueous formulations. These results demonstrate that the non-aqueous formulations induce a satisfactory immune response to the vaccinating antigen that is equivalent to that induced by the commercial, aqueous vaccine.

EXAMPLE 2

Further variations of the non-aqueous formulations then were tested.. The data presented in this example show that the non-aqueous formulations may be successfully used to deliver antigen, adjuvant, and an anti-parasitic chemical. In addition, the release profile of the antiparasitic compound was not altered compared to the release profile observed using a standard vehicle for administering the compound.. These novel, combined formulations accordingly act both as a tick vaccine (similar to TickGARD Plus) and an injectable abamectin formulation (similar to "Avomec," MSD Agvet).

Serum abamectin levels and the Bm86 antibody titers, generated by eight experimental combined formulations, were compared to the control products

TickGARD Plus and Avomec. The stability of abamectin in the formulations after storage for eight weeks at 40° C also was investigated. None of the formulations tested contained water, and all utilized DMSO to introduce the Bm86 and Quil A (vaccine components) into the respective vehicles containing abamectin.

The DMSO/Bm86/adjuvant phase was pre-prepared., as was the

GF/PG/ Abamectin phase. Formulations containing abamectin at 1% w/v contained Bm86 at 12.5 μg/ml and QuilA at 0.25mg/ml. Formulations containing abamectin at 2% w/v contained Bm86 at 25μg/ml and QuilA at 0.5mg/ml.

Injections were administered to each animal on Days 1 and 29.

Depending on abamectin concentration, experimental formulations were administered to cattle at a rate of lml/ 100kg or 2ml/ 100kg (to give 200 μg abamectin/kg body weight). TickGARD Plus was administered as in Example 1.

Avomec was administered at a dose of 2ml/100kg (to give 200μg abamectin kg).

In order to use the same animals as controls for both reference products,

TickGARD Plus and Avomec were administered to the same individuals on opposite sides of the neck. The excipients used in differing proportions in the experimental formulations were: glycerol formal (GF) and propylene glycol

(PG). Adjuvants used were Quil A, Montanide 52 and Pluronic Polymer L121.

Table 3: Formulations

Formulation Composition (per lOOmL, volumes are rounded)

(% abamectin cone w/v)

1: (1%) Standard commercial TickGARD Plus and Avomec

Dosage: 2ml/100kg body wt.

Avomec /

2ml animal TickGARD Plus

2: (2%)

Dosage: lml/lOOkg body wt. PG/GF: 58ml propylene glycol, 38ml glycerol formal,

GF/PG/DMSO 2000mg abamectin

DMSO: 6ml DMSO, 1.43mg Bm86, 8.33mg Quil A

3: (1%)

Dosage: 2ml 100kg body wt. PG/GF: 58ml propylene glycol, 39ml glycerol formal,

GF/PG/DMSO lOOOmg abamectin

DMSO: 3ml DMSO. 1.43mg Bm86, 8.33mg Quil A

4: (2%) lml Surfadone/lOOkg Surfadone: 94ml glycerol formal, 2000mg abamectin

DMSO: 6ml DMSO, 1.43mg Bm86, 8.33mg Quil A

5: (1%)

Dosage: 2ml/100kg bodv wt. PG/GF: 48ml propylene glycol, 32ml glycerol formal,

GF/PG/OIL/L121/DMSO lOOOmg abamectin

L121 5.0ml

Marcol 52 12.5ml

DMSO: 3ml DMSO, 1.43mg Bm86, 8.33mg Quil A

6: (1%)

Dosage: 2rru7100kg bodv wt. PG/GF: 48ml propylene glycol, 32ml glycerol formal,

GF/PG/OIL/Tween/DMSO lOOOmg abamectin

Tween 5ml

Marcol 52 12.5ml

DMSO: 3ml DMSO. 1.43mg Bm86, 8.33mg Quil A

7: (1%)

Dosage: 2πύ7100kg body wt. PG/GF: 55ml propylene glycol, 37ml glycerol formal,

GF/PG/OIL/L 121/DMSO lOOOmg abamectin

L121 5ml

DMSO: 3ml DMSO. 1.43mg Bm86. 8.33mg Quil A

8: (1%)

Dosage: 2ml/ 100kg bodv wt. GF: 97ml glycerol formal, lOOOmg abamectin

GF/DMSO DMSO: 3ml DMSO. 1.43mg Bm86. 8.33mg Quil A

9: (1%)

Dosage: 2.06ml/100kg body Avomec: 97ml Avomec (lOmg/ml abamectin) wt. DMSO: 3ml DMSO. 1.43mg Bm86. 8.33mg Quil A

Avomec/DMSO 1. Abamectin stability and safety

After 6 weeks' storage at 40°C there was evidence of significant abamectin losses in two formulations (50% loss in the Formulation 4 (Surfadone) and 10% loss in Formulation 6 (oil + L121). Abamectin in all other experimental formulations was stable. No formulations caused adverse systemic effects.

Serum anti-Bm86 titers

These results demonstrate that the novel formulations can readily deliver antigen and adjuvant in an immunologically active state. When used to prime and boost naive (not previously immunized) cattle the novel formulations stimulated antibodies to the vaccinating antigen at levels which were not significantly different to the commercial vaccine TickGARD Plus. Table 5: Average serum abamectin concentration (ng/ml)

The foregoing results indicate that non-aqueous formulations of the present invention can deliver abamectin effectively. Two important parameters warranting consideration, when analyzing the abamectin release profile, are the maximum serum abamectin level and the area under the curve. Two experimental formulations (1% Abamectin in 40:60 GF/PG + vaccine in DMSO and 1% Abamectin in GF + vaccine in DMSO) produced satisfactory abamectin release profile (90%) area under the curve of the reference product (Avomec) and peak serum levels 74% and 71% of Avomec respectively). Introduction of mineral oil or the pluronic polymer L-121 (both good immunological adjuvants) into such formulations appeared to reduce somewhat the bioavailability of abamectin.

These results, together with the results of the antibody titers generated from the same formulations, show that the novel formulations described can be used effectively to deliver antigen, adjuvant and antiparasitic chemicals to animals. In particular, formulations containing Abamectin at 1%> w/v in either Glycerol Formal alone or a 60:40 mix of Glycerol Formal and Propylene Glycol and the vaccine components Bm86 and QuilA dissolved in DMSO can deliver abamectin with a bioavailability comparable to the reference product Avomec and generate anti-Bm86 antibodies in a manner comparable to the reference product TickGARD Plus. Of the two, the 60:40 mix is probably preferable for economic reasons. Abamectin in these formulations is as stable as it is in Avomec following storage for 6 weeks at 40°C. This application claims priority to Australian Provisional Applications

PO7791 and PO7792, filed July 10, 1997, the disclosures of which are hereby incorporated by reference in their entirety. Those applications describe treatment of animals with non-aqueous vaccine formulations comprising an anthelminitic and an immunogenic polypeptide together with an adjuvant in a polar non- aqueous solvent. Animals treated with the vaccines developed strong antibody responses against the immunogenic polypeptide.

Claims

WHAT IS CLAIMED IS:

1. A single-phase, non-aqueous vaccine composition comprising an antigenic polypeptide in a polar non-aqueous solvent.

2. A composition according to claim 1, wherein said non-aqueous solvent comprises dimethyl sulfoxide..

3. A composition according to claim 2, further comprising a non- aqueous hydroxylic solvent.

4. A composition according to claim 3, wherein said hydroxylic solvent is selected from the group consisting of glycerol formal and propylene glycol.

5. A composition according to claim 3, wherein said hydroxylic solvent comprises a mixture of glycerol formal and propylene glycol.

6. A composition according to claim 3, wherein dimethyl sulfoxide comprises up to about 6%> v/v of said non-aqueous solvent.

7. A composition according to claim 4 wherein the volume ratio of glycerol formal to propylene glycol is about 40:60.

8. A composition according to claim 7, wherein dimethyl sulfoxide comprises about 3-6%> of said non-aqueous solvent.

9. A composition according to claim 8, wherein dimethyl sulfoxide comprises about 3%> of said non-aqueous solvent.

10. A composition according to claim 1, further comprising at least one non-aqueous adjuvant.

11. A composition according to claim 10, wherein said adjuvant is selected from the group consisting of saponins, mineral or non-mineral oils, detergents, and pluronic polymers.

12. A composition according to claim 11, wherein said adjuvant is Marcol 52, and comprises up to about 12.5% v/v of said non-aqueous solvent.

13. A composition according to claim 11, wherein said adjuvant is Pluronic Polymer, and comprises up to about 5%> v/v of said non-aqueous solvent.

14. A composition according to claim 11, wherein said adjuvant comprises a mixture of (i) Marcol 52, comprising up to about 12.5% v/v of said non-aqueous solvent, and (ii) Pluronic Polymer, comprising up to about 5% v/v of said non-aqueous solvent.

15. A method of manufacturing a single-phase non-aqueous vaccine formulation according to claim 1, comprising admixing an immunogenic polypeptide with a polar, non-aqueous solvent.

16. A method of vaccinating a mammal, comprising administering to said mammal an effective dose of a vaccine according to claim 1.

17. A single-phase, non-aqueous pharmaceutical formulation comprising an immunogenic polypeptide and a pharmaceutical in a polar non- aqueous solvent.

18. A formulation according to claim 17, wherein said pharmaceutical is an endectocide.

19. A formulation according to claim 18, wherein said endectocide is an anthelmintic.

20. A formulation according to claim 19, wherein said anthelmintic is a macro cyclic lactone of the milbemycin or avermectin group of anthelmintics.

21. A formulation according to claim 20, wherein said anthelmintic is abamectin or ivermectin.

22. A method of manufacturing a non-aqueous formulation according to claim 17, comprising admixing an immunogenic polypeptide and a pharmaceutical in a polar non-aqueous solvent.

23. A method of treating or preventing a disease in a mammal, comprising administering to said mammal an effective dose of a formulation according to claim 17.

24. A single-phase, non-aqueous vaccine formulation prepared by admixing an immunogenic polypeptide with a polar non-aqueous solvent.

25. A single-phase non-aqueous vaccine formulation prepared by admixing an immunogenic polypeptide and a pharmaceutical with a polar non- aqueous solvent.

26. A formulation according to claim 1, where said immunogenic polypeptide is derived from a disease-causing organism.

27. A formulation according to claim 26, wherein said disease-causing organism is a parasitic organism.

28. A formulation according to claim 27, wherein said immunogenic polypeptide is derived from an organism selected from the group consisting of, nematodes and insects.

29. A formulation according to claim 28, wherein said insect is a tick selected from the group consisting of Boophilus spp, Haemaphysalis spp, Otobius spp, Rhiphicephalus spp, Amblyomma spp, Dermacentor spp, Ixodes spp, and Hyakmma spp.

30. A formulation according to claim 29, wherein said tick is selected from the group consisting of Boophilus microplus, Boophilus annulatus, Boophilus decoloratus, Otobius megnini, Rhiphicephalus appendiculatus, Dermacentor andersoni, Dermacentor variabilis, Haemaphysalis longcornis, Amblyomma variegatum and Ixodes holocydus).

31. A formulation according to claim 1 wherein said immunogenic polypeptide is Bm86.

32. A formulation according to claim 28, wherein said insect is a haematophageous parasitic insect selected from the group consisting of Haemotobia spp, Hypoderma spp, Dermatobia spp, Anopheles spp, Lucilia spp, Ctenocephalides spp, Chrysomya spp, Gasterophulus spp, Culicoides spp, Stomoxys spp, Linognathus spp, Solenoptes spp, Haematopinus spp, Melophagus spp, Aedes spp, and Culez spp.

33. A formulation according to claim 28, wherein said parasite is a nematode selected from the group consisting of Strongylus spp, Trichostrongylus spp, Haemonchus spp, Ostertagia spp, Ascaris spp, Trichinella spp, Toxascaris spp, Uncinaria spp, Ancylostoma spp, Trichuris spp, Dirofilaria spp, Necator spp, Ancylostoma spp, Ascaris spp, Trichuris spp, Enterobius spp, Strongyloides spp, and Wuchereria spp.

34. A formulation according to claim 33, wherein said nematode is selected from the group consisting of Strongylus vulgaris, Trichostrongylus colubriformis, Haemonchus contortus, Ostartagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonna, Uncinaria stenocephala, Ancylostoma caninum, Trichuris vulpis, Dirofilaria immitis, Necator americanus, Ancylostoma duodenale, Asceris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides ster corals and Wuchereria bancrofti.