WO1998018453A1

WO1998018453A1 - Oral vaccines for young animals with an enteric coating

Info

Publication number: WO1998018453A1
Application number: PCT/IB1997/001136
Authority: WO
Inventors: Jay Dean Gerber
Original assignee: Pfizer Inc.
Priority date: 1996-10-28
Filing date: 1997-09-22
Publication date: 1998-05-07
Also published as: AU4133397A; ZA979592B; AR010031A1

Abstract

The present invention provides a vaccine formulation and method for oral vaccination of animals of weaning age or younger against a pathogen in the presence of interfering maternal antibodies. The formulation of the invention comprises an antigen in an enteric coating.

Description

ORAL VACCINES FOR YOUNG ANIMALS WITH AN ENTERIC COATING

FIELD OF THE INVENTION

The present invention relates to compositions and methods for oral vaccination of animals of weaning age or younger against disease

BACKGROUND OF THE INVENTION

Vaccination of animals of weaning age or younger against a disease-causing pathogen is often compromised by the presence of interfering maternal antibodies, i e , maternally-derived antibodies that are directed against the pathogen For example, canine parvovirus (CPV), canine distemper virus (CDV), canine coronavirus (CCV) and canine rotavirus (CRV) diseases are the most important diseases in dogs of weaning-age or younger However, vaccination of these young dogs against these viral pathogens is compromised by the presence of the interfering maternal antibodies This creates a "window of susceptibility" to infection in the young animal because the maternal antibody titer in the animal is often high enough to interfere with vaccination, but too low to provide an adequate level of protection Parenterally-injected vaccines are of less than optimal efficacy in the^e young animals, and must generally be administered multiple times to ensure adequate immunity In addition, parenterally-injected vaccines often do not induce immunity until at least several weeks after weaning, depending upon the titer of the interfering maternal antibodies

Since the entry points for many pathogens, including CPV, CDV, CCV and CRV, are through the oral and nasal routes, oral immunization may trigger a more rapid and robust immune response against one or more of these pathogens by eliciting the production of secretory immunoglobulin A (IgA) antibodies at mucosal sites Such an immune response is triggered when an antigen is presented to the epithelium of gut-associated lymphoid tissues, such as Peyer's patches See, for example, O'Hagan, 1992, Clin Pharmacokinet 22 1 -10, Caldwell et al , 1982, J Pharm Pharmacol 34 520, and U S Pat No 5,075, 109 However, oral immunization has generally remained underexploited as a method of vaccination because major problems remain over how to deliver an oral vaccine to the gut mucosal tissue without significant degradation in the digestive tract (O'Hagan, 1992, above)

Compositions and methods have been described for the oral immunization of humans and various animal species For example, U S Pat No 5,352,448 discloses an oral vaccine formulation for ruminants, comprising an antigen composition in a delivery vehicle consisting of a water-swellable hydrogel matrix, which allows for delivery of the antigen to mucosa-associated lymphoid tissue in the post-ruminal portion of the digestive tract U S Pat No 5,176,909 discloses compositions for oral administration to humans or animals, comprising an immunogen, gelatin of a particular molecular weight range, and an enteric coating U S Pat No 5,075,109 discloses a method for targeting a bioactive agent, e g , an antigen, to the Peyer's patches by microencapsulating the agent in a biocompatible polymer or copolymer, such as poly(DL-lactιde-co-glycolιde) U S Pat No 5,032,405 discloses an oral formulation, comprising a lyophilized mixture of a biologically active agent, e g , an immunogen, in combination with maltose, a particulate diluent, and a coating comprising an alkaline-soluble polymeric film U S Pat No 4,152,415 discloses a method of immunizing field-raised swine against dysentery, comprising administering a sequential series of parenteral and enteric - coated oral preparations of a virulent isolate of killed cells of Treponema hyodysentenae

Despite these disclosures, it is unclear whether an oral vaccine formulation can trigger a sufficiently robust immune response in animals of weaning-age or younger to overcome the presence of interfering maternal antibodies

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an oral vaccine formulation for administration to an animal of weaning age or younger, which comprises an immunologically effective amount of an antigen in an enteric coating, and that it is capable of triggering a protective immune response against a pathogen in the presence of interfering maternal antibodies The protective immune response can be triggered in animals at a younger age than can be obtained with conventional parenteral vaccines In a second aspect, the invention provides a method for vaccinating an animal of weaning age or younger against a disease, comprising orally administering to the animal a vaccine comprising an immunologically effective amount of an antigen in an enteric coating, which vaccine is capable of triggering a protective immune response against the disease, even in the presence of interfesing maternal antibodies In a non-limiting embodiment, the animal is a dog of weaning age or younger, although the invention is generally applicable to other animals of weaning age or younger, including cats, cows, pigs, sheep, and horses, among others Other features and advantages of the invention will be described in the following sections of the specification

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an oral vaccine against a pathogen for administration to an animal of weaning age or younger, which vaccine comprises an immunologically effective amount of an antigen prepared from the pathogen in an enteric coating, that it is capable of triggering a protective immune response against the pathogen in the presence of interfering maternal antibodies The vaccine may be administered to any animal of weaning age or younger of a particular species, whether or not interfering maternal antibodies are present The vaccine of the invention may be formulated for administration to any one of various species of animals, including dogs, cats, cows, pigs, sheep, and horses, among others In a preferred but non-limiting embodiment, the animal is a dog of weaning age or younger In a further non-limiting embodiment, the pathogen is a canine virus The enteric coating protects the antigen from the low pH environment of the stomach, but dissolves and releases the antigen in the region of gut-associated lymphoid tissues As a result, a gut mucosal immune response is triggered in the animal that is sufficiently robust to overcome the interference of maternal antibodies and protect the animal against disease caused by the pathogen The vaccine of the invention may comprise an antigen prepared from any pathogen to which the animal is susceptible In a non-limiting embodiment, the antigen is prepared from a viral pathogen For example, for dogs, the antigen may be prepared from CRV, CPV, CDV or CCV However, antigen may alternatively be prepared from other pathogenic viruses, or from pathogenic bacteria, protozoans, or fungi that cause disease in the particular animal species Vaccine antigens which are currently administered parenterally to the animal species, as known in the art, may be also used in the oral vaccine formulation For oral vaccination of a feline species, the antigen may be prepared from any pathogen that causes disease in cats including, for example, calici virus, feπne herpes virus, leucopenia virus, feline coronavirus including feline infectious peritonitis (FIP) virus, non-FIP coronavirus, and rabies virus, among others

For oral vaccination of a bovine species, the antigen may be prepared from any pathogen that causes disease in cows including, for example, bovine herpes virus, bovine respiratory syncitial virus, bovine diarrhea virus, parainfluenza type 3 virus, bovine coronavirus, bovine rotavirus, rabies virus, and E coll, among others For oral vaccination of a swine species, the antigen may be prepared from any pathogen that causes disease in pigs, including, for example, swine parvovirus, swine pseudorabies virus, rabies virus, transmissible gastroenteritis virus, swine rotavirus, and virulent E coll, among others

The antigen may comprise any component of a viral or cellular pathogen, which component is capable of triggering a protective immune response in the animal in the presence of interfering maternal antibodies Such a component includes, but is not limited to, whole viral particles or cells, viral capsids and envelopes, whole membrane preparations and membrane fractions, proteins, peptides, glycoproteins, exotoxins, endotoxins, enzymes, and antigenic subunits of any of the aforementioned components Also included is a DNA sequence which encodes an antigenic peptide or protein Smaller antigenic components may be derived from larger components such as membranes and proteins by fractionation or degradation techniques known in the art Once obtained, the antigen may be modified using standard techniques, for example, by adding or substituting chemical substituents that alter rates of degradation in vivo or that increase antigenicity The antigen may also be further processed, such as by lyophihzation, according to standard techniques

Pathogenic strains of viral particles or cells for use to prepare antigen can be obtained using standard isolation techniques from fluids, tissues or organs of infected animals exhibiting clinical symptoms of a particular disease Alternative sources of pathogen include publicly available deposits, such as those stored at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852-1776, USA

Where whole live viral particles or cells of a pathogen are used as antigen, they are preferably modified to an attenuated form, or are inactivated Methods of attenuating a pathogen are known in the art and include, for example, serial passaging of the pathogen For example, viral pathogens may be serially passaged in an in vitro culture of susceptible cells, preferably mammalian cells, and most preferably in cells of the particular animal species to be vaccinated For example, a canine viral pathogen can be serially cultured in canine kidney cells according to known techniques, such as those described in Appel ef al , 1979, Vet Records 105 156-159, U S Pat No 4,193,991 , and U S Pat No 4,303,645, which are incorporated herein by reference Other methods of attenuation include, but are not limited to, exposure of a pathogen to a mutagenic agent, e g , a chemical mutagen or ultraviolet radiation A pathogen may also be attenuated by knocking out one or more essential metabolic genes, for example, by use of recombinant DNA techniques which are known in the art, including by homologous recombination Alternatively, a pathogen can be inactivated, such as by exposure to formaldehyde, glutaraldehyde or ultraviolet light, among other inactivating agents After the attenuation step, viral particles or cells exhibiting sufficient attenuation of pathogenicity compared to the parental strain are preferably selected and clonally propagated according to known tissue culture techniques Attenuation may be indicated, for example, by the appearance of a novel temperature sensitivity or a novel auxotrophy, or a reduction in a virulence trait, such as infectivity or the severity or rate of progression of a symptom or condition associated with the disease, among other indications For example, attenuation of a pathogen may be indicated by the appearance of a novel temperature sensitivity in which the attenuated cells will not grow, or will grow only at a significantly reduced rate compared to the pathogenic parental strain, at the normal body temperature of the animal to be vaccinated

The oral vaccine formulation of the invention comprises an immunologically effective amount of antigen, which is that amount of antigen capable of triggering a protective immune response in an animal of weaning age or younger in the presence of interfering maternal antibodies As used herein, a "protective immune response" is defined as any immunological response, based either on antibody or cell-mediated immunity, or both, occurring in the animal after administration of the vaccine of the invention, which response either prevents or reduces infection by the pathogen, or eliminates or reduces the severity, or slows the rate of progression, of one or more clinical symptoms or conditions normally associated with a disease caused by the pathogen As used herein, "interfering maternal antibodies" refer to those antibodies present in the young animal that originate from a maternal source, are directed against a pathogen, and are present in sufficient titer to otherwise detectably reduce, or interfere with, the efficacy of a parenterally administered vaccine directed against the pathogen For example, a titer of interfering maternal antibodies of about 1 16 or higher will interfere with successful immunization The oral vaccine of the invention is formulated so that the antigen is protected from degradation in the low pH environment of the stomach, thus allowing the antigen to be delivered to the region of the gut necessary to stimulate a mucosal immune response that is sufficient to trigger a protective immune response, even in the presence of interfering maternal antibodies To accomplish this, the oral vaccine formulation comprises an immunologically effective amount of antigen protected within an enteric coating that resists dissolution in the low pH environment of the stomach, but which readily dissolves to release the antigen only at about pH 6 or higher The enteric coating is any type of substance useful to coat the antigen that meets these requirements Materials and methods to produce enteric coatings are known in the art For example, a material useful to produce an enteric coating may be a polymethacrylic acid copolymer, one non-limiting example of which is a partially methylated polymethacrylic acid copolymer such as EUDRAGIT S100^® type B, NF or EUDRAGIT L100^® (Rohm Tech Inc , Maiden, MA ) The pH at which the enteric coating dissolves can be specifically adjusted or broadened as needed, for example, by adjusting the concentration of the enteric coating component applied, or by combining one or more enteric coating components together For example, EUDRAGIT S100^® may be combined in varying ratios with EUDRAGIT L^® to produce a coating that dissolves at a specifically modified pH Such a technique is described in U S Pat No 5,032,405 which is incorporated herein by reference The thickness of the enteric coating may also be adjusted to help control the timing and location of dissolution of the formulation

The oral vaccine formulation of the invention may further comprise one or more veterinaπly-acceptable excipients known in the art, such as those that improve preparation, handling and stability of the formulation Non-limiting examples of such excipients include, for example, lactose, sucrose, maltose, mannitol, starch, gums, gelatin, cellulose derivatives, magnesium salts such as magnesium stearate, magnesium carbonate, and magnesium oxide, calcium salts such as calcium carbonate and calcium sulfate, silica gel, and talc, among others The oral vaccine formulation may optionally comprise other useful components, including, for example, an undercoating, or a colorful dye

One non-limiting example of an oral vaccine formulation comprises the following components antigen, lactose, magnesium stearate, modified cellulose gum, an undercoating, and an enteric coating Various modifications of this formulation will be immediately apparent to those skilled in the fields of immunology, veterinary medicine, delivery system formulations, etc Suitable other vehicles and additives are known, or will be apparent to those of skill in the art See, e g , Remington's Pharmaceutical Science, 18th Ed , 1990, Mack Publishing, which is incorporated herein by reference The ingredients of the vaccine are combined according to known techniques to produce an orally administrable formulation Thus, after preparing and mixing the antigen and any excipients, the mixture is compressed to an administrable form such as a tablet, pill, or capsule For example, the formulation may be compressed using a Carver Lab Press (Fred S Carver Inc , Menomee Falls, Wl ) The formulation is then provided with an enteric coating such as by the Open-Pan Ladle Coating Process or the Wurster Coating Process, which are well-known in the art The present invention further provides a method for vaccinating animals of weaning age or younger against a pathogen, comprising orally administering to the animal a vaccine capable of triggering a protective immune response against the pathogen, even in the presence of interfering maternal antibodies, said vaccine comprising an immunologically effective amount of antigen in an enteric coating that dissolves only at about pH 6 0 or higher In a preferred, but non-limiting, embodiment, the vaccine formulation is administered to a dog of weaning age or younger to protect against canine disease The canine disease may be caused by any pathogen that infects dogs, including but not limited to CRV, CPV, CDV or CCV

An effective dosage of the oral vaccine formulation may be determined by conventional means, starting with a low dose of the formulation and then increasing the dosage while monitoring the effects, and systematically varying the dose as well Numerous factors may be taken into consideration when determining an optimal dose per animal Primary among these is the breed, size, age, and general condition of the animal to be vaccinated Other considerations include the presence and titer of interfering maternal antibodies in the animal, the presence of other drugs, and the like The actual dose is preferably chosen after consideration of the results of other animal studies

In a preferred embodiment, an immunologically effective amount of vaccine comprising a viral pathogen will comprise from about 10³ to about 10⁹ TCID₅₀ per animal, more preferably the amount will be from about 10" to about 10⁸ TCID₅₀ per animal, and most preferably the amount will be from about 10^{5 5} to about 10⁷ TCID₅₀ per animal In a preferred embodiment, an effective dose range of bacteria will comprise from about 10⁵ to about 10¹² plaque-forming units (pfu) per animal

Vaccine regimens may also be selected based on the above-described factors Thus, animals may be vaccinated soon after birth, or just prior to, at the time of, or soon after weaning, depending upon analysis of these factors

Supplemental administrations, or boosters, may be required for full protection One method of detecting whether adequate immune protection has been achieved is IO determine seroconversion and antibody titer in the young animal after vaccination A benefit of the vaccine of the present invention is that a protective immune response can be triggered in animals at a younger age than can generally be obtained with conventional parenteral vaccines For example, in dogs, whereas conventional parenteral vaccines often do not induce immunity prior to 12 to 16 weeks of age depending on maternal antibody titer, the oral vaccine of the invention can induce immunity at 4-6 weeks of age Thus, in dogs, a preferred, though non- limiting, administration schedule is a single oral dose of the vaccine at the time of weaning / e , at approximately 4 to 8 weeks of age Alternatively, two oral doses may be necessary if an adequate immune response does not occur in a dog after an initial dose, or if the dog has an interfering maternal antibody titer in excess of about 1/128

The present invention further provides combination vaccines, comprising an immunologically effective amount of a first antigen capable of triggering a protective immune response against a pathogen in the presence of maternal antibodies, and an immunologically effective amount of one or more other antigens that can trigger a protective immune response against the same or a difference pathogen

The following specific embodiments of the invention are intended to be illustrative and not limiting

EXAMPLE 1: ORAL VACCINE PREPARATION

A general oral formulation was prepared as follows A ratio by weight of 60 parts lactose (direct compression grade) (Sheffield Products, Norwich, N Y ), I part AC-DI-SOL^® modified cellulose gum (FMC Corp , Philadelphia, PA ), 1 part magnesium stearate (impalpable powder) (Mallinckrodt, St Louis, MO ), and 4 parts lyophi zed canine parvovirus that had been attenuated by serial passage in dog kidney cells, were used to make a tablet formulation The lactose, modified cellulose gum and lyophihzed vaccine were mixed together The magnesium stearate was added to produce a final mixture, which was compressed into tablets using a Carver Lab Press (Fred S Carver Inc , Menomee Falls, Wl ) with a one-half inch tooling dye at 5,000 pounds/sq inch The tablets were rotated in a pan coater during application of OPADRY CLEAR^™ undercoating (Colorcon, West Point, PA ) An enteric coating was then added by applying EUDRAGIT S100^® methacrylic acid copolymer type B, NF (Rohm Tech Inc , Maiden, MA ) to a final concentration of about 3% by weight using the Open-Pan Ladle Coating Process

EXAMPLE 2: EFFICACY TESTING OF AN ORAL FORMULATION WITHOUT AN ENTERIC COATING

Attenuated CPV, CCV and CRV were grown separately in cultures of canine kidney cells to titers of 10^{6 7} TCID₅₀ of CPV, 10^{5 3} TCID₅₀ of CCV, and 10^{8 2} TCID₅₀ of

CRV, respectively, according to standard tissue culture techniques The attenuated viral particles were lyophihzed and formulated together into tablets comprising 10^{6 3} TCID₅₀ of CPV, 10^{4 3} TCID₅₀ of CCV, and 10^{7 5} TCID₅₀ of CRV, and containing all of the components listed above in Example 1 , but without an enteric coating

Four 3 5- to 5 5-month-old SPF beagles, which were seronegative for anti- CPV antibodies, but seropositive for anti-CCV and CRV antibody titers, were each administered a single oral dose of non-enteric coated vaccine The dogs were bled once a week for 6 weeks and serum tested for anti-CPV, CCV and CRV antibodies Response to vaccination was inconsistent (Table 1 ) Only two of the four dogs seroconverted to CPV (1/64 and 1/512, respectively, at 14 days post- vaccination, and 1/2,048 and 1/4,096, respectively, at 42 days post-vaccination) For both CCV and CRV, one out of four dogs showed an increase in titer following vaccination The results were equivocal, possibly due to a failure to consistently deliver the antigen intact to the intestine

TABLE 1

RESPONSE TO NON-ENTERIC COATED ORAL FORMULATION

EXAMPLE 3: EFFICACY TESTING OF AN ORAL FORMULATION WITH AN ENTERIC COATING

Attenuated CPV (10" TCID₅₀) and CCV (10^J ' TCID₅₀) were formulated together into tablets as above in Example 2, but with an enteric coating, comprising about 3% (v/v) EUDRAGIT S100^®, that dissolves only at about pH 6 0 or higher Twenty 7-week-old SPF beagles, seronegative for CPV and CCV were distributed into two groups of 10 dogs each, taking litter distribution into consideration The ten dogs in the first group (vaccinates) (Table 2) each received a single dose of the vaccine by placing a single tablet in the back of the mouth and gently pushing it into the throat for the dog to swallow The ten dogs in the second group (controls) were not vaccinated (Table 3) Serum samples were collected prior to vaccination and for 5 weeks post-vaccination for CPV virus neutralization testing and CCV ELISA

Since the dogs seroconverted to CCV prior to vaccination, indicating exposure to CCV, the efficacy of the CCV fraction of the vaccine could not be determined However, the efficacy of the CPV fraction of the vaccine compared to the control was established, as shown in Tables 2 and 3 According to those results, the oral vaccine with the enteric coating elicited a rapid and strong protective immune response in all ten of the vaccinates (Table 2) All ten non- vaccinated control dogs failed to develop CPV serum neutralizing antibody titers (Table 3) This result is in contrast to that obtained with oral vaccine lacking an enteric coating, as demonstrated above in Example 2 (Table 1)

Observations were made regarding vaccine safety Vaccinates were observed daily for 5 days post-vaccination for signs of vomiting, depression and diarrhea In addition, temperatures were taken daily for 5 days post-vaccination, and blood collected once a week for CBC, acute-phase protein, and immune cell marker analyses None of the vaccinates showed any signs of vomiting, diarrhea, depression, or a febrile response Furthermore, none of the vaccinates showed any alpha-1 glycoprotein abnormalities, abnormal immune cell marker profiles, or abnormal blood profiles Thus, the vaccine appears to be safe

TABLE 2

CPV SERUM NEUTRALIZATION TITERS OF VACCINATED DOGS LACKING MATERNAL ANTI-CPV ANTIBODIES^*

A serum neutralizing antibody titer equal to or greater than 1 16 is considered sufficient to protect against virulent CPV challenge (see 9 C F R § 113 317) a = Geometrical mean titer

TABLE 3 CPV SERUM NEUTRALIZATION TITERS OF CONTROL DOGS

EXAMPLE 4: RESPONSE OF DOGS WITH MATERNAL ANTI-CPV ANTIBODIES TO ORAL CPV VACCINATION

Twenty 7-week-old beagles with maternal anti-CPV antibodies were divided into two groups, taking litter, sex and maternal antibody titer into consideration Ten dogs in the first group (vaccinates) (Table 4) received one dose of the enteric- coated vaccine prepared as above Ten dogs in the second group (controls) (Table 5) received no vaccine A single tablet was placed in the back of the mouth of each vaccinate, and gently pushed into the throat for the dogs to swallow

The data in Tables 4 and 5 demonstrate that the enteric-coated oral vaccine formulation of the invention immunizes dogs of weaning age against CPV infection in the presence of maternal anti-CPV antibody titers of up to 1 128 Out of four dogs with interfering maternal antibody titers of 1 128 (BGZ, BKY, BLY, CYZ), three (BGZ, BKY, BLY) developed strong serum-neutralizing antibody titers after a single oral dose of the vaccine A second dose may be required for dogs having interfering maternal antibody titers greater than 1 128

None of the vaccinates showed a febrile response or any evidence of vomiting, depression or diarrhea post-vaccination, again indicating that the vaccine

TABLE 4

CPV SERUM NEUTRALIZATION TITERS OF VACCINATED DOGS HAVING MATERNAL ANTI-CPV ANTIBODIES

TABLE 5

CPV SERUM NEUTRALIZATION TITERS OF CONTROL DOGS HAVING MATERNAL ANTI-CPV ANTIBODY TITERS

All patents, patent applications, and publications cited above are incorporated herein by reference in their entirety

The present invention is not to be limited in scope by the specific embodiments described, which are intended as single illustrations of individual aspects of the invention Functionally equivalent compositions and methods are within the scope of the invention Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the fields of immunology, veterinary medicine, delivery system formulations, and related fields, from the foregoing description and accompanying drawings Such modifications are intended to fall within the scope of the appended claims

Claims

CLAIM

1 An oral vaccine capable of triggering a protective immune response against a pathogen in an animal of weaning age or younger in the presence of interfering maternal antibodies, said vaccine comprising an immunologically effective amount of an antigen prepared from the pathogen in an enteric coating that dissolves only at about pH 6 0 or higher

2 The oral vaccine of claim 1 , wherein the antigen is prepared from a canine pathogen selected from the group consisting of canine parvovirus, canine distemper virus, canine coronavirus, and canine rotavirus

3 The oral vaccine of claim 1 , wherein the antigen is prepared from canine parvovirus

4 The oral vaccine of claim 3, wherein the antigen is attenuated canine parvovirus

5 The oral vaccine of claim 1 , further comprising one or more excipients selected from the group consisting of lactose, sucrose, maltose, mannitol, starch, gum, gelatin, a cellulose derivative, magnesium stearate, magnesium carbonate, magnesium oxide, calcium carbonate, calcium sulfate, silica gel, and talc

6 The oral vaccine of claim 1 , further comprising lactose, magnesium stearate, modified cellulose gum, and an undercoating, and wherein the enteric coat comprises a partially methylated polymethacrylic acid copolymer

7 The oral vaccine of claim 6, wherein the partially methylated polymethacrylic acid copolymer is EUDRAGIT S100^® polymethacrylic acid copolymer, type B, NF

8 A method for vaccinating an animal of weaning age or younger against a pathogen, comprising orally administering to the animal a vaccine capable of triggeπng a protective immune response against the pathogen in the presence of interfering maternal antibodies, said vaccine comprising an immunologically effective amount of an antigen prepared from the pathogen in an enteric coating that dissolves only at about pH 6 0 or higher

9 The method of claim 8, wherein the animal is a dog

10 The method of claim 9, wherein the antigen is prepared from a pathogen selected from the group consisting of canine parvovirus, canine distemper virus, canine coronavirus, and canine rotavirus

1 1 The method of claim 9, wherein the antigen is prepared from canine parvovirus

12 The method of claim 11 , wherein the antigen is attenuated canine parvovirus

13 The method of claim 8, wherein the formulation further comprises one or more excipients selected from the group consisting of lactose, sucrose, maltose, mannitol, a gum, gelatin, a cellulose derivative, magnesium stearate, magnesium carbonate, magnesium oxide, calcium carbonate, calcium sulfate, silica gel, starch, and talc

14 The method of claim 8, wherein the formulation further comprises lactose, magnesium stearate, modified cellulose gum, and an undercoating, and wherein the enteric coat comprises a partially methylated polymethacrylic acid copolymer

15 The method of claim 14, wherein the partially methylated polymethacrylic acid copolymer is EUDRAGIT S100^® polymethacrylic acid copolymer, type B, NF 16 The method of claim 9, wherein the interfering maternal antibodies are present in the dog's serum in a titer of about 1 16 or higher