WO1993004698A1

WO1993004698A1 - Method of enhancing anti-rabies immune responses

Info

Publication number: WO1993004698A1
Application number: PCT/US1992/007509
Authority: WO
Inventors: Keith N. Haffer
Original assignee: Smithkline Beecham Corporation
Priority date: 1991-08-29
Filing date: 1992-08-28
Publication date: 1993-03-18
Also published as: AU2682592A

Abstract

This invention provides a method of enhancing an animal's immune response to a selected vaccine, e.g., a rabies vaccine, by coadministering the selected vaccine and a Feline Leukemia Virus vaccine.

Description

METHOD OF ENHANCING ANTI-RABIES IMMUNE RESPONSES

Field of the Invention

This invention relates generally to methods of enhancing animal immune responses to selected vaccines.

Background of the Invention

Rabies is a centuries old disease, endemic in almost all countries, which has devastating clinical symptoms. Control of the disease continues to be very difficult, particularly in view of the extensive reserve of the virus in wild type animals and the continual encroachment of human settlements on the habitat of these animals. The increased proximity between these infected wild animals, humans, and domesticated animals provides a very real threat of further spread of this disease.

While there are several vaccines which are effective in preventing the clinical manifestation of rabies following infection with the virus, these vaccines are expensive and unsuitable for large scale preventative use.

For example, M. Lafon et al, Vaccine , 6_:362-8 (1988) describes immunity against the European bat rabies virus DUV3 induced by human rabies vaccines prepared with the rabies laboratory strains Pitman-Moore (PM) , Pasteur

SUBSTITUTESHEET virus (PV4) and Flury LEP (LEP) . All the vaccines protected against challenge with Challenge virus standard, but only the PV vaccine protected mice against a DUV3 challenge. Two studies comparing antibody titers and protection from clinical symptoms and death resulting from infection with various pathogens show that there is a direct relationship between antibody titers and rabies death rates. [See, e.g., T. Bunn et al, "The Relationship Between Rabies Antibody Titers in Dogs and Protection from Challenge", in Rabies Information Exchange., p.43-45 (June, 1983); Kihm et al, Co p. Immun. Microbiol. Infect. Pis., 5_:227-232 (1982)]. Therefore, a vaccine capable of enhancing antibody titers following vaccination with a rabies vaccine would be beneficial in enhancing protection against death caused by rabies infection.

There remains a need in the art for effective compositions and methods for effectively vaccinating animals against rabies infection, and having utility in the large-scale prevention of rabies.

Summary of the Invention

As one aspect the present invention provides a method of enhancing the immune response in non-human animals, particularly cats, to a selected antigen by the

SUBSTITUTE SHEET simultaneous or sequential administration of a Feline Leukemia Virus (FeLV) vaccine and the selected antigen.

In one embodiment the selected antigen may be a first FeLV vaccine, wherein a second vaccine administered simultaneously or sequentially therewith is either the same or a second, different FeLV vaccine formulation. In a preferred embodiment, the second vaccine is a rabies virus vaccine, to which immunity is enhanced by the concurrent administration of a FeLV vaccine. In another aspect, the present invention provides a method for enhancing the immune response in non-human animals, particularly cats, to more than a single selected vaccine by the simultaneous or sequential administration of a Feline Leukemia Virus vaccine, a rabies vaccine and one or more selected vaccines.

As still a further aspect, the invention provides a vaccine composition comprising an effective amount of a feline leukemia vaccine and an effective amount of a rabies vaccine. Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments of the present invention.

SUBSTITUTESHEET Brief Description of the Drawings

Fig. 1 illustrates the results of the rabies GMT Rapid Fluorescent Focus Inhibition Test (RFFIT) , in virus neutralization (VN) titer, performed on cat sera after simultaneous administration of Rabies V vaccine and one of our FeLV vaccines at three and five weeks post inoculatio .

Fig. 2 illustrates the results of the rabies GMT RFFIT, in International Units, performed on cat sera after simultaneous administration of a vaccine and one of four FeLV vaccines at three and five weeks post inoculation.

Detailed Description of the Invention

It has been surprisingly discovered that the administration of an effective amount of a Feline Leukemia Virus (FeLV) vaccine in combination, sequentially or simultaneously, with an effective amount of one or more selected vaccines provides a method for enhancing the immune system in cats. A number of commercially available vaccines exist against FeLV, which virus is normally immunosupressive in part. These vaccines may be useful in the present invention. A suitable feline leukemia vaccine for use in the method of this invention includes an inactivated feline leukemia vaccine described in U.S.

SUBSTITUTESHEET Patent No. 4,332,793, which is non-infectious, immunogenic and non-immunosuppressive. The aforementioned patent is incorporated by reference for the purpose of providing a description of this FeLV vaccine, which contains strains KT-A, B, and C of FeLV and is adjuvanted with Quil A and Al(OH)₃. A modification of this vaccine (Leukocell 2, SmithKline Beecham) , characterized by enhanced immunogenicity may also be used in this method. In the modified vaccine, the antigenic content is increased and a different inactivating agent is used so that cats can effectively be protected with two primary doses, rather than three [see, Haffer et al, Vaccine. _3:12-16 (1990)].

Other suitable feline leukemia vaccines may be used in the method of the invention. One commercial supplier of such vaccines is Bio-Trends International Inc. , whose vaccine consists of whole virions of the feline retrovirus in a concentrated antigenic mass. The virus particles are selectively inactivated by a procedure which destroys the nucleic acid core without affecting or denaturing the antigenic proteins which produce the serum virus neutralizing antibodies. Other suitable commercially available feline leukemia vaccines include Fel-O-Vax Lv-K, which contains a killed virus [Fort Dodge Laboratories, Fort Dodge, Iowa], Leukocell

SUBSTITUTESHEET and Leukocell 2 [Norden Laboratories, Lincoln, NE] , VacSYN/FeLV [Synbiotics Corp. , San Diego, CA] , and Covenant [Diamond Laboratories] .

According to this invention the administration of an effective amount of an FeLV vaccine to a cat provides a general, non-specific immune system enhancement which can operate to further stimulate the animal's immune response to other antigens co- administered therewith. For use herein, "co- administration" includes administration of the FeLV vaccine in combination with one or more other selected vaccines, the administration being simultaneous (as in a single composition) or sequential (separate FeLV and other vaccine compositions administered at separate times in various sequences) . When used in this method to enhance the immune response of the animal to a second selected vaccine, an effective amount of a FeLV vaccine is defined as the amount of immunogen(s) plus adjuvant which will afford greater than 72% protection to vaccinated cats which have been challenged with virulent FeLV. For example, an effective amount of Leukocell vaccine ranges from about 3000 to about 20,000 relative units (R.P.). Effective amounts of other available FeLV vaccines are readily available to one of skill in the art.

SUBSTITUTE SHEET Vaccines to which an enhanced immunological response is expected upon co-administration with a FeLV vaccine include, among others, rabies, feline panleukopenia, rhinotracheitis, calici, and chlamydial vaccines. In this invention, any vaccine containing antigens from one or more of the above viruses or other viruses which are recognized by the immune system of the cat, are useful. Among commercially available vaccines which may be used, include, without limitation, Felocell CVR-C (SmithKline) , Fel-O-Vax (Ft. Dodge) and Eclipse 4

(Solvay) which are directed against feline panleukopenia, rhinotracheitis, calici and chlamydia psittaci.

The effective amount of the non-FeLV vaccine or antigen depends upon the particular vaccinal agent selected, but is generally the same as the amount of that non-FeLV vaccine when administered as a sole vaccinal agent. Thus, for example, an effective amount of a rabies vaccine for use in this method ranges from about 0.8 to about 3.0 I.U. or relative potency (R.P.). The determination of the appropriate dosages or effective amounts is within the skill of the art, given this teaching.

As described in the examples below, an illustrative example of the method of this invention involves the co-administration of a rabies vaccine with a

SUBSTITUTE SHEET FeLV vaccine. Vaccination of cats according to the present invention with rabies vaccines followed by vaccination with feline leukemia virus vaccines resulted in an enhanced anti-rabies immune response as measured by standardized methodologies. The rabies antibody titers were higher those generated by vaccination with the rabies vaccine only. In addition, rabies titers in cats receiving FeLV vaccines reached a maximum two weeks earlier than cats receiving only rabies vaccine. The invention encompasses a combined formulation containing both rabies vaccine components and feline leukemia vaccine components and a method of administering both vaccine components simultaneously. The inclusion of the feline leukemia vaccine components in a single composition, or for simultaneous introduction in the animal may permit a reduction of the immunogenic amount of rabies vaccine components in a single vaccine composition. However, if the feline leukemia vaccine and rabies vaccine are administered separately, usually at two different sites, the amount of rabies vaccine components would probably not be reduced.

The present invention also encompasses a method of co-administering more than one selected vaccine with the FeLV vaccine, or alternatively co-administering one or more selected vaccines with both the FeLV and rabies vaccines to enhance the animal's immune response to the

SUBSTITUTESHEET rabies vaccine as well as to the third and other vaccines. These additional vaccines include, among others, feline panleukopenia, rhinotracheitis, calici, and chlamydial vaccines. Effective amounts of these vaccines for co-administration are desirably equivalent to the amounts normally administered when such vaccines are administered alone.

Ordinarily, the administration protocol of the feline leukemia vaccine parallels that of the other vaccine being administered, to which an enhanced immune response is desired. At present, the subcutaneous route is preferred, with a 1 - 2 mL dose of each vaccine antigen. Either antigen may be administered first. Preferably, all vaccine components are administered in liquid form.

A preferred embodiment of the method of the invention includes administering (intramuscularly or subcutaneously) an aqueous suspension or solution containing one or more of mLV, feline panleukopenia, calici, rhinotracheitis, or chlamydia vaccines reconstituted with a rabies virus at one site. Reconstitution involves mixing freeze dried vaccine with liquid rabies. The FeLV vaccine is administered separately at a separate site.

SUBSTITUTESHEET Also provided by the invention is a pharmaceutical vaccine composition comprising an effective amount of an FeLV vaccine and an effective amount of another virus vaccine in admixture with a nontoxic and sterile pharmaceutically acceptable carrier. One such pharmaceutical composition includes an effective vaccinal amount of FeLV antigens and an effective vaccinal amount of rabies virus antigens. Another composition includes an effective amount of FeLV and an effective vaccinal amount of one or more of the antigens of rabies virus, feline panleukemia, rhinotracheitus, calicivirus and chlamydia.

Suitable carriers are well known to those of skill in the art. However, exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil and water. Additionally, the carrier or diluent may include a time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax.

Additionally, the vaccine components (FeLV and rabies) may be admixed or adsorbed with a conventional, adjuvant. The adjuvant is used as a non-specific irritant to attract or enhance an immune response. Such adjuvants include, among others, amphigen, aluminum hydroxide, muramyl dipeptide and saponins such as Quil A.

SUBSTITUTE SHEET This vaccine composition may be used in the methods of this invention to protect the animal against feline leukemia and rabies, or used with other antigens to protect the animal against other pathogens. The compositions of the present invention are advantageously made up in a dosage unit form adapted for the desired mode of administration. The quantity of FeLV antigen and rabies and/or other viral antigens forming the active ingredients in each dosage unit will be such that one, or possibly more, units containing between 3000 to 20,000 R.U. (relative units) FeLV and >1.0 R.P. (relative potency) rabies or other virus are required for each therapeutic administration. The composition may be buffered at physiological pH, in a form ready for injection.

Dosage and administration protocol can be optimized in accordance with standard vaccination practices. Typically, the vaccine will be administered subcutaneously, although other routes of administration may be used, such as intramuscular, oral, intraocular, intradermal and intranasal administration. It is expected that a useful single dosage for average mature felines is in the range of 1 to 200 micrograms of FeLV antigen, preferably 8 - 30 micrograms, in admixture with the same or lesser amount of the rabies antigen and optional adjuvant. It will be understood, however, that the specific dose level for any particular animal will depend upon a variety of factors including the number and identity of vaccinal antigens to be coadministered with this composition, the age, general health, sex, and diet of the animal; the species of the animal; the time of administration; the route of administration; interactions with any other drugs being administered; and the degree of protection being sought. The vaccine(s) can be administered initially in late summer or early fall and can be readministered two to six weeks later, if desirable, or periodically as immunity wanes, for example, every two to five years. Of course, as stated above, the administration can be repeated at suitable intervals if necessary or desirable.

The examples which follow are illustrative only and not intended to limit the scope of the present invention.

Example 1 - Preparation of a Selected Rabies Vaccine Baby Hamster Kidney cells (BHK-21, NVSL Lot 6, passage 10) are seeded in growth medium at 1.2 - 4.0 x 10⁴ cells per cm². The growth medium may be either DMEM medium or Optime medium supplemented with 10% Tryptose phosphate broth (3% final concentration, w/v) , bovine serum, 10 mM Hepes buffer and gentamicin within the specified limits of 9 CFR 114.10. Approximately 24 hours post-culture initiations, the cells are infected. The growth medium is removed and Rabies Strain PV-Paris, SVR-289, the seed virus, (0.01 to 0.8 MOI) [Dr. George Baer, Center for Disease Control, Atlanta, GA] is adsorbed onto cells for one hour at approximately 35°C-37°C.

The PV-Paris strain of fixed rabies virus was obtained by the Pasteur Institute from the Pan American Zoonoses Center (CEPANZO) , Buenos Aires, Argentina (PV plO) n 1965. After 1 intracerebral passage on young rabbits, the virus was termed PV-Paris pass.11. After an undetermined number of passages on BHK-21 cells, the virus was concentrated, purified and kept frozen. After adsorption, the volume of medium was adjusted to approximately 250 mL for 850 cm² bottles and proportionately to 100 L for increased surface bottles. The roller bottles were incubated at approximately 35°C - 37°C for 3 - 4 days after infection. After two additional passages, the virus pool had a titer of 7.0 x 10⁸*PFU/mL.

The virus has been characterized with neutralizing anti-glycoprotein monoclonal antibodies. It is identical to PV-CEPANZO virus, and different from PV- 11 HD virus [Pitman Moore strain] and PV-13 BOB virus [WHO/RAB.RES./84/20].

SUBSTITUTESHEET At 3 - 4 days post-infection, roller bottles are removed from incubation and examined microscopically for contamination. Following examination, the growth culture is aseptically decanted into sterile containers. Cultures are then refed daily with production media and harvested daily for the next 4 days.

When harvesting, following microscopic examination, fluid is decanted from roller bottles into sterile collection vessels. Culture vessels may be refed with maintenance medium. The vessels may be frozen or refrigerated prior to further processing and harvest containing growth and production medium may be pooled. Only harvest materials found to be sterile are used in the vaccine. The minimum acceptable titer or harvest material is 10⁷-⁵ FAID₅₀ mL. Samples of harvested material are pooled and tested for mycoplasma prior to inactivation.

The virus is inactivated as follows. The virus-laden fluids are transferred into suitable containers and beta propiolactone (BPL) is added at a ratio of 1:4000. Inactivation is effected at 4°C for at least 96 hours.

Following inactivation, representative samples are taken from the pool and tested for completion of inactivation in accordance with 9 CFR 113.209(d) (2) (i) or as follows. The chemically-treated virus-laden fluids are inoculated onto monolayers of BHK-21 cells.

SUBSTITUTESHEET Production medium is DMEM or Optimem growth medium supplemented with serum (concentration 0-1%) or gamma irradiated BSA (0.1% - 0.5%). After 14 - 21 days of incubation at 35°C to 37°C, including at least one subculture, cell cultures are stained with specific fluorescein-labeled antiserum. The absence of virus- infected cells indicates a satisfactory inactivation test.

Following inactivation, merthiolate [Thimersol, N.F.] is added as a preservative to a final concentration of <1:10,000. The inactivated product is stored at about 4°C until it is assembled.

Pooled inactivated viral fluids (1960 mL) were mixed with DMEM + 0.5% FBS (980 mL) to effect a 1.5 dilution (1 part harvest + 0.5 parts medium) . The 2700 mL of diluted harvest was mixed with 300 mL of aluminum hydroxide adjuvant [2% A1₂0₃] to a final concentration of 10% v/v and mixed overnight at 4°C. The final bulk vaccine contains 90% standardized rabies fluids and 10% aluminum hydroxide adjuvant by volume.

The product is standardized by pre-inactivation titer. An acceptable titer is 10⁷³ FAID₅₀/ml prior to adding the adjuvant. Harvests may be diluted with production media to attain standardized concentration of antigen. These bulk virus fluids may be concentrated 2 - 20x by sterile ultrafiltration.

SUBSTITUTESHEET Each 1 mL dose contains at least 10⁷-²⁵ FAID₅₀ (pre-inactivation titer) of rabies virus.

Example 2 - Rabies Virus Challenge Studies

The vaccine of the invention is prepared as described above and had a relative potency of 0.96, measured at 0, 1, 3, 6, 9 and 12 months post vaccination by RFFIT, and a preinactivation titer of 10⁷-⁴ FAID₅₀/mL.

Two hundred and thirty-one (231) SPF cats [Liberty Labs] were randomly assigned to three groups (1) immunogenicity vaccine (n=136) ; (2) non-vaccinated controls, (n-56) , and Imrab [a rabies vaccine available from Institut Merieux] (n=39) . Thirty (30) subcutaneous vaccinates, 10 Imrab vaccinates and 10 non-vaccinated controls were challenged at one year post-vaccination with 1 mL NYC Georgia street rabies virus, F-83-1 (0.5 mL per masseter muscle) according to 9 CFR 113.129(b) (3) (IV) . Back titrations of the challenge material in mice yielded 916,016.2 MLD₅₀ per mL of inoculum. All 10 non-vaccinated controls and 1 subcutaneous vaccinee died during the 90 day observation period following challenge. All cats were confirmed as being rabies positive by fluorescent antibody. A monoclonal antibody to the nucleoprotein (NC protein) conjugated to fluorescein isothiocyanate (FITC) was

SUBSTITUTESHξET obtained from the Center for Disease Control, Atlanta, Georgia. The FA staining is performed as described in Goldwasser and Kissling, Proc. Soc. Exp. Biol. , 18:219 (1958) with conjugate prepared according to standard procedures and treated twice with normal BHK-21/S13 cells. The wells are examined with a fluorescent microscope using suitable exciter and barrier filters. These results indicate that the rabies vaccine afforded 96.7% protection against a rabies challenge which killed 100% of the susceptible, age-matched controls.

Example 3 - Rabies/Feline Leukemia Vaccine Protocol

Three FeLV vaccines described below were administered concurrently with an inactivated rabies vaccine (designated Rabies V) , prepared as described above in Example 1, to determine the effects of the FeLV fractions on the cats' responses to rabies antigen. Rabies titer as determined by RFFIT, described below, were determined at the time of vaccination and at 3 and five weeks post vaccination by standardized methods as described in Smith et al, "A rapid tissue culture test for determining rabies neutralizing antibody", in Laboratory Techniques in Rabies (eds. M. Kaplan and H. Koprowski) , W.H.O. , Geneva 1973, p. 354-360.

SUBSTITUTE SHEET Thirty SPF cats [Liberty Labs, Liberty, NJ] were housed in isolation units throughout the tests. The cats were 10-14 weeks old at the time of first vaccination. All cats were initially vaccinated with Rabies

V. Ten cats only received the rabies vaccine (controls) while the other 20 were separated into 4 treatment groups of 5 cats each. Cats in these treatment groups were vaccinated according to manufacturer's route of administration at a site distinct from where the rabies vaccine was administered.

The three FeLV vaccines were as follows. The Covenant formulation [Diamond Scientific] contains bovine serum, FeLV strain and is adjuvanted with oil. VacSyn [Synbiotics] contains bovine serum, FeLV strains KT-A,B, and C. Fel-O-Vax [Ft. Dodge] contains bovine serum and FeLV strain A and is adjuvanted with oil.

The Covenant vaccine was administered intramuscularly, while all others were administered subcutaneously. At three weeks post first vaccination, cats in the treatment groups received the second dose of their respective FeLV vaccine.

Cats were initially prebled then bled again at 3 and 5 weeks post rabies vaccination. Sera was collected in vacutainer tubes and allowed to clot. The separated sera was analyzed in the RFFIT test along with

SUBSTITUTE SHEET a U.S. Rabies serum standard [available from the Centers for Disease Control, Atlanta, Georgia USA upon request] .

Each vial of the U.S. standard rabies immune globulin contains 59 International Units (IU) of rabies antibody. Each vial is reconstituted with 10. mL of sterile distilled water and then rinsed 2x with 0.5 L of sterile distilled water. A pool of two reconstituted vials is prepared and diluted to 2 IU/mL. Diluted globulin is aliquoted in small volumes and stored at - 20°C until used. The RFFIT described in Smith et al, cited above, was modified to be performed in 96-well microtiter plates.

All sera are inactivated at 56°C for 30 minutes. The sera is then treated with kaolin to adsorb non-specific inhibitors. An equal volume of 25% kaolin is added to each serum, and the mixture is allowed to stand at room temperature for 20 minutes with frequent agitation. It is then centrifuged at 800 g for 10 minutes at room temperature. The supernatant liquid is considered a 1:2 dilution of the original serum.

Dilutions are made in a 96-well microtiter plate. 0.1 mL of the proper dilutions of challenge virus is added to 0.1 L final volumes of all serum dilutions. The chambers are then incubated at 37°C in a controlled humidity carbon dioxide chamber for 90 minutes.

SUBSTITUTE SHEET The rabies challenge virus standard [CVS; ATCC No. VR959] is propagated in BHK-21 cells. The optimal challenge dose is the highest dilution of virus that gives strong FA staining in the cells after 24 hours. BHK-21 cultures are grown in plastic containers in DMEM and supplemented with 10% of inactivated fetal calf serum and 10% of tryptose phosphate broth. BHK monolayers 1-4 days old (between the 25 and 50th passage level) are trypsinized for use in the RFFIT. Immediately before their use in the RFFIT, the cells are treated with a solution of DEAE-dextran [Pharmacia, Uppsala, Sweden] in growth medium (lOμg/mL) for 10 minutes at room temperature. Suspensions containing 5xl0⁵ cells/0.05 mL of growth medium are added to each of the wells and the plate is then returned to the carbon dioxide chamber.

The growth medium is removed after a 24-hour incubation period, and the plates are then rinsed once in phosphate-buffered saline, once in acetone at room temperature, and then fixed for 30 minutes in acetone at 22°C.

The results, illustrated below in Table I, Fig. 1 and Fig. 2 are expressed as virus neutralization (VN) titer and International Units (I.U.). The VN titer represents the serum endpoint; I.U. is the VN test serum divided by reference serum. The analysis comparing treatment groups to the control group were determined by

SUBSTITUTESHEET Student's t test. A reduction of 50% or more in the number of fields with fluorescing cells indicates the presence of neutralizing antibody in the test serum.

As shown in the Table I below, all rabies/FeLV groups demonstrated a maximal rise in titer at 3 weeks and then a decline at 5 weeks. Conversely, the control titers rise at 3 weeks and maximize at 5 weeks. At the 3 week bleed, a statistically significant difference exists between the rabies/Covenant and the control group (p<0.02).

The Covenant vaccine has the strongest adjuvant and could promote higher titers to coadministered vaccines. VacSyn has no adjuvant and Fel-O-Vax has an adjuvant of lesser quality than the first two vaccines. However, all coadministered vaccines boosted titers to rabies. The FeLV antigens, with or without an additional adjuvant, appear to act as adjuvants or immune enhancing agents.

The administration of FeLV antigens concurrently with inactivated rabies vaccine also acts to boost initial rabies titer above that which is anticipated by rabies vaccination alone.

SUBSTITUTESHEET

SUBSTITUTE SHEET

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modification and alterations are believed to be encompassed in the scope of the claims appended hereto.

SUBSTI UTE SHEET

Claims

What is claimed is:

1. A method of enhancing an animal's immune response to a selected vaccine comprising administering simultaneously or sequentially an effective amount of a Feline Leukemia Virus vaccine and an effective amount of said selected vaccine.

2. The method of claim 1 wherein said selected vaccine is selected from the group comprising a rabies virus vaccine, a feline panleukopenia vaccine, a feline rhinotracheitis vaccine, a feline calici vaccine, and a feline chlamydial vaccine.

3. The method according to claim 2 wherein the selected vaccine is a rabies vaccine.

4. The method according to claim 1 further comprising the step of administering a second Feline

Leukemia Virus vaccine two to six weeks after the first vaccination.

5. The method according to claim 3 wherein the second Feline Leukemia Virus vaccine is administered three weeks after the first vaccination.

SUBSTITUTE SHEET

6. A method of enhancing an animal's immune response to a selected vaccine comprising administering simultaneously or sequentially a Feline Leukemia Virus vaccine, a rabies virus vaccine and said selected vaccine.

7. The method according to claim 6 wherein the selected vaccine is selected from the group comprising Feline panleukopenia, rhinotracheitis, calici, and chlamydial vaccines.

8. A pharmaceutical vaccine composition comprising an effective amount of a Feline Leukemia Virus vaccine and an effective amount of another selected viral vaccine in combination with a pharmaceutically acceptable carrier.

9. The composition according to claim 8 wherein the selected vaccine is selected from the group comprising Feline panleukopenia, rhinotracheitis, calici, and chlamydial vaccines.

10. The composition according to claim 8 wherein said selected vaccine is a rabies virus vaccine.

SUBSTITUTE SHEET