HIGH POTENCY VACCINES

Abstract: The invention provides a method for preparing a vaccine comprising combining both killed and modified-live forms of an infectious agent in an effective combination. When used to vaccinate an animal, the combined vaccine provides an improved immune response, both in terms of duration and extent of the response, and is particularly useful in the veterinary field. The invention further relates to a high-potency vaccine prepared by the method of the invention.

Description: HIGH POTENCY VACCINES TECHNICAL FIELD The present invention relates to the vaccination of animals for the purpose of raising an immune response and enhancing protection from infection and/or disease. In particular, the present invention relates to formulations, e.g. , vaccines, and methods suitable for use in the vaccination of animals. BACKGROUND OF THE INVENTION Animals that are capable of raising an immune response to antigens, and particularly agricultural animals such as cattle, swine, horses, poultry and the like, as well as humans, can often be temporarily or permanently immunized against viral infections by administering to the animal either a killed form of the virus, or a live, but attenuated, form of the virus. The immune system of the animal responds by generating cell-mediated or antibody-mediated responses, or both, to infectious agents. The overall level of immunity that is developed in the animal is, in part, a function of the cell-mediated and antibody- mediated responses generated by the animal's immune system. See, generally, Immunology. Chapter 11 - "Cell-Mediated Immunity", Roitt, et al. eds., C.V. Mosby Co., St. Louis, Mo. (1985), the disclosure of which is incorporated herein by reference. To confer resistance to various diseases, a polyvalent vaccine can be used, containing response-inducing forms of several different infectious agents. A common polyvalent vaccine for immunizing cattle, for instance, includes infectious bovine rhinotracheitis virus, bovine diarrhea virus, parainfluenza-3 virus, bovine respiratory syncytial virus, and various bacteria. It is believed that there may be physiological limitations to the ability of an animal to generate an immune response to injected antigens. The administration of a polyvalent vaccine may, therefore, provide a lower level of immunity to some or all of the individual viruses, as compared to the level achievable by vaccination with any single agent alone. On the other hand, the immune response (as determined by the antibody-mediated and cell-mediated responses ) to a given antigen can typically only be increased a limited extent by increasing the amounts of the antigen administered. At higher amounts, the administration of larger doses may not produce a corresponding increase in effective immunity. It is important, however, for a vaccine to produce extensive and complete immunity, i.e., one in which appropriate levels of both antibody-mediated, as well as cell- mediated, parameters are achieved. A recurring concern with both attenuated virus and killed virus vaccines is the fact that many present vaccines, and particularly veterinary vaccines, are not able to generate immune responses that are consistently as complete and/or prolonged as would be desired, particularly when used to vaccinate animals that are either highly stressed or very young. SUMMARY OF THE INVENTION The present invention provides a vaccine, and a method of vaccinating an animal using the vaccine, that provides an improved immune response, both in terms of duration and extent of the response, and is particularly useful in the veterinary field, for the vaccination of animals that are highly stressed or very young. The method of the present invention, for immunizing an animal against an infectious agent, comprises the step of administering to the animal an effective combination of both killed and modified-live (i.e., attenuated) forms of the infectious agent. The invention provides a vaccine comprising both killed and attenuated infectious agent. In one embodiment, the vaccine of the present invention is provided as a single, stable formulation, e.g. , in which both forms are provided together in a vehicle suitable for administration to an animal. In another embodiment, the vaccine is provided as a kit, comprising separate formulations of killed infectious agent and modified-live infectious agent forms, both formulations being suitable for concurrent administration to an animal in order to achieve an effective combination. The word "concurrent", as used in this sense, refers to routes and/or times of administration suitable to achieve an improved immune response of the sort described in the present specification. This invention is based upon the discovery that the administration of two or more viral forms according to the method of the present invention can achieve enhanced immune responses, i.e., in terms of an optimal combination of the animal's cell-mediated and antibody-mediated responses. The improvement in immunity, e.g., the antibody- response, can be surprisingly high, and often is many-fold greater than the antibody response produced through the administration of either of the viral forms alone. DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides a method of immunizing an animal against an infectious agent comprising the step of administering to the animal an effective combination of both killed (i.e., inactivated) and modified-live (i.e., attenuated) forms of the agent. The invention is applicable to a wide variety of infectious agents, including in bovine, infectious bovine rhinotracheitis (IBR) virus, bovine virus diarrhea (BVD) virus and parainfluenza-3 (PI-3) virus, among others. Representative vaccines (and presently available vaccine forms) for the vaccination of humans include the diphtheria-tetanus- pertussis (DPT: toxoid-toxoid-killed bacteria) vaccine, measles-mumps-rubella (MMR: all attenuated viruses) vaccine, smallpox (attenuated virus), yellow fever (attenuated virus), poliomyelitis (attenuated types I, II and HI), rabies (killed virus), Rocky Mountain Spotted Fever (killed rickettsial organisms), tuberculosis (attenuated mycobacteria), typhoid (killed bacteria), cholera (killed bacteria), influenza (killed virus), meningococcus (polysaccharide vaccine), plague (killed bacteria), and pneumococcus (multivalent) vaccines. The term "infectious agent", as used herein refers to any agent capable of causing disease in an animal, and capable of being provided in both the killed form and the modified live form, as defined more fully below. The term will frequently be used interchangeably herein with the preferred form of infectious agent, i.e., viruses, however it is understood that forms other than viruses are contemplated as well, e.g., bacteria and rickettsial organisms. By "killed" infectious agent, reference herein is made to an agent that has been inactivated, e.g., chemically treated, so as to render the agent non-infective, that is, unable to either cause disease or infect cells in the vaccinated animal, or therefore, to reproduce itself. Chemical inactivation can be accomplished by a variety of means within the skill of those in the art, e.g., by the use of aldehydes, lactones, imines, and the like. Included within the definition of "killed" agents are those prepared by "classic" approaches such as: killed whole pathogens; toxoids derived from pathogens; purified surface components; and conjugated surface components; as well as those prepared by "molecular" approaches, such as recombinant-derived proteins; synthetic peptides; and anti-idiotype 10 antibodies. (See, e.g., S. Plotkin and E. Mortimer, eds. , "New Technologies for Making Vaccines", pp. 568-575, in Vaccines. W.B. Saunders Company, 1988. By "modified-live infectious agent", reference is made to an agent that has been attenuated, e.g., processed or otherwise treated so as to render it capable of growth in the vaccinated animal but non-virulent, i.e. , incapable of causing disease. Included within the definition of "modified-live" agents are those prepared by "classic" approaches such as: being modified by passage in cell culture; variant viruses from other species; temperature-selected mutants; and reassorted genomes; as well as those prepared by more recent "molecular" approaches, such as DNA modification mutants, and recombinant viruses. (See, e.g. , S. Plotkin and E. Mortimer, eds., "New Technologies for Making Vaccines", pp. 568-575, in Vaccines. W.B. Saunders Company, 1988. The amount of infectious agent administered to an animal in accordance with the invention can be expressed both in terms of the absolute amount of each form, and the relative amounts of the forms. In terms of the former, it is preferred that each form be present in an amount that alone would be effective in achieving an immune response, e.g., the amount of killed virus should be at least the amount necessary to pass the minimum potency test for such vaccine, for the species to be vaccinated. Similarly, the amount of modified-live form should be sufficient to pass a minimum titer necessary for achieving the potency requirement. Both determinations can be made using standard techniques familiar to those skilled in the art. See, e.g., "Immunodeficiency Diseases", T.J. Fischer, pp. 345-364, in Manual of Allergy and Immunology. G. Lawlor, et al. , Eds., Little, Brown & Company, Boston, MA (1984). The relative amounts of the viral forms in a preferred vaccine of the present invention can be readily determined by those skilled in the art in view of the present teaching. Preferably, the relative amounts will be those amounts which, when administered, will provide optimal immunity, e.g. , amounts capable of increasing the antibody titer obtained by at least 20 % , preferably by at least 40 % , and most preferably by at least 50% or more over the titer obtainable using either form alone. Thus, for example, an antibody titer produced by administration of one virus form (either killed or modified-live virus) might be 1 :40, and with a combination of that form plus the other virus form, the titer might be 1:50; the combined viral forms thus providing a 25% titer increase. The titer increase, expressed as a percentage as described in the present specification, including Examples, is calculated as follows: (post-vaccination titer) - (pre-vaccination titer) titer increase = X 100 (pre-vaccination titer) While it is desirable to administer a viral quantity that is sufficient to result in substantial antibody production in the animal, those skilled in the art will appreciate that care should be taken to not employ excessive amounts of either viral form, in order to avoid overwhelming the ability of the body to develop effective immunity to the virus and also to other infectious agents. In preparing a vaccine of the invention, it is preferable that most of the virus particles be killed, with only a relatively small percentage being of the modified-live form. In a typical preferred embodiment, for instance, it has been found that the optimal number of modified-live virus particles per dose is at least 16,000. However, it is preferred to use about 4,000,000 or more infectious particle equivalents, i.e., killed virus particles, per dose. Thus, if the preferred minimum numbers of both the modified-live virus particles and the killed virus particles are used, a total of about 4,016,000 virus particles will be present in each dose, i.e., with about 16,000 (equal to about 0.4%) being of the modified-live form. It should be understood tL _: the above amounts are preferred minimums and the amount of either or both forms of virus particles per dose may be increased over these minimums. Accordingly, the relative proportions of killed and modified-live virus forms in the vaccine may vary substantially. It is preferred that an effective vaccine of the invention should have between about 0.1 % and about 1 %, preferably between about 0.3 % and about 0.7%, and most preferably between about 0.4% and about 0.6%, of the total number of virus particles in the modified-live form. Vaccines of the present invention can be prepared by any suitable means. See, e.g., Plotkin, and E. Mortimer, eds., "New Technologies for Making Vaccines", pp. 568-575, in Vaccines. W.B. Saunders Company, 1988. A vaccine of the invention can include killed and modified-live forms of more than one infectious agent, thereby providing a polyvalent vaccine of such combined vaccines. In addition to the killed and modified-live virus forms noted above, a vaccine of the invention can include additional components as well. Such components desirably include pharmaceutically acceptable carriers and/or adjuvants as are known in the art; for instance, aluminum hydroxide has been found to work quite well as a carrier/adjuvant in field trials. Adjuvants suitable for the particular virus being treated may be used as well; for instance, in the case of infectious bovine rhinotracheitis and bovine virus diarrhea vaccines, oil-type and polymer adjuvants are particularly useful. A vaccine of the invention can also include a stabilizer for increasing the shelf life of the vaccine. Such stabilizers are known in the art and are generally expected to be compatible with the vaccines of the present invention. The present invention is particularly effective for a variety of bovine viruses. Additionally, a vaccine of the invention is expected to yield similar results in any animal, including mammals and poultry, e.g., in sheep, pigs, dogs, cats, and horses, as well as in chickens or turkeys. The invention may be more readily understood by reference to the following non- limiting EXAMPLES. EXAMPLES EXAMPLE 1 Infectious Bovine Rhinotracheitis (TBR) A group of 25 young, weaned beef calves, each weighing 300-500 pounds (135- 225kg) was divided into three groups of six animals and one group of seven animals. The first three groups were vaccinated with a chemically-inactivated (i.e., killed) IBR virus ("KV", available from Sanofi Animal Health, Inc., Lenexa, KS, as List No. 6047), modified-live IBR virus ("MLV", available from Sanofi animal health as List No. 2636), or a killed/modified-live virus combination ("KV/MLV") IBR vaccine, respectively. The combined vaccine was prepared in this case by using the already-liquid KV formulation as a diluent to rehydrate the lyophilized MLV preparation. The vaccines were prepared so as to provide comparable quantities of each antigen form in each cattle dose. The fourth group was not vaccinated and served as a control group. Animals in the vaccinated groups were randomly further divided into two subgroups and were injected either intramuscularly ("IM") or subcutaneously ("SQ") with a single dose. Serum samples were drawn before vaccination and again 35 days later. Sera were assayed by a standard serum neutralization test for antibodies against IBR. The mean antibody titers of sera collected at day 35 were as follows: Titer Group I MLV vaccine 1:25 Group II KV vaccine 1:59 Group m MLV/K V vaccine 1:121 Control No vaccine no increase in titers The data show that simultaneous injection of one dose of the combined KV/MLV vaccine of the invention resulted in a titer 105 % greater than that of the killed virus vaccine alone and 375 % greater than that of the modified-live virus vaccine alone. EXAMPLE 2 Dual Dose Vaccine A second group of 29 calves was divided into four groups as in Example 1 and given a two dose vaccination regimen (the doses separated by 21 days), instead of the one dose IBR vaccinations described in Example 1, keeping the same quantities per dose. The calves within each group were randomly divided into groups receiving either HVI or SQ injections. The control group was the same as for Example 1, as were the sources and preparation of the vaccines themselves. The mean IBR antibody titers were as follows: The results were similar to Example 1 in that the combined KV/MLV vaccine of the invention provided a 173 % increase in mean titer over the MLV vaccine alone and 121 % increase over the KV vaccine alone. EXAMPLE 3 Comparison with Commercial Vaccines A group of 20 young beef calves was divided into five groups of four calves each and vaccinated intramuscularly with the combined MLV/KV vaccine in the same manner as Example 2, or with commercially available IBR vaccines having either killed or modified-live viral forms (according to the vaccination regimen recommended by the manufacturer). Mean IBR titers alter vaccination were compared to the four commercially available vaccines. The commercial vaccines were administered according to instructions on the package label. Mean antibody titers to IBR are given below: Titer MLV/KV vaccine (Example 1) 97 Sanofi Animal Health, Inc. List No. 6047 (KV) 41 Smithkline Beecham, "Cattlemaster" (MLV) 43 Diamond, "Horizon" vaccine (MLV) 34 Anchor, "Discover" vaccine (KV) 26 It can be seen that the combined modified-live/killed virus vaccine of the invention gave increases of 136%, 125%, 182% and 279% over commercial vaccines 1 through 4, respectively. EXAMPLE 4 Dual Dose Vaccine In this study, a group of 144 very young Holstein dairy calves, each weighing 120-500 pounds (55-225 kg), was divided into three groups and were then vaccinated intramuscularly with a two dose regimen of MLV, KV, or MLV/KV vaccines as in Example 2. A fourth group of twelve animals was not vaccinated and served as a control group. Two vaccine doses were administered about 21 days apart. Pre-vaccination titers ranged from 1:2 to 1:128, which is characteristic of maternal antibody levels in this type of young animal. Mean post- vaccination titers to IBR for each group of calves were as follows: The increase of the antibody titer for a vaccine of the invention was 29.3 % over the KV vaccine alone and 206 % over the MLV vaccine alone. Of interest in this study was the finding that only (10%) of the animals given the combined MLV/KV vaccine did not respond with at least an eight-fold increase of titer, while 20% of the calves given KV alone and 27.7% of the calves given MLV alone failed to respond with at least an eight- fold increase in antibody titer. EXAMPLE 5 Timing of Administration A group of young beef calves was vaccinated as in Example 1 , i.e., using one dose of the MLV/KV vaccine. A second group of ten animals was vaccinated with IBR (MLV) and then re-vaccinated with IBR (KV) two weeks later. Serum was collected about fourteen days later on all animals. Mean serum titers for the two groups were as follows: The data suggest that the combination of modified-live and killed virus forms, given simultaneously, yields significantly higher mean antibody titers than separate, nonconcurrent doses of modified-live virus and killed virus given 14 days apart. EXAMPLE 6 Bovine Virus Diarrhea (BVD) A group of 135 young Holstein calves each weighing 150-400 pounds (55 to 180 kg) was divided into three groups and vaccinated with modified-live vaccine (Sanofi Animal Health, Inc. , List No. 2639) or a killed vaccine (Sanofi Animal Health, Inc., List No. 5692), or a combined MLV/KV bovine virus diarrhea vaccine prepared by using the KV as a diluent for reconstituting the dehydrated MLV vaccine. Two doses of the respective vaccines were administered 21 days apart. Pre-vaccination and post- vaccination sera were collected and assayed for BVD antibody titers by a standard neutralization test. Pre-vaccination titers ranged from less than 1:2 to 1: 1024. Average post-vaccination titer increases for the groups of test animals were as follows: Titer Increase MLV vaccine 1: 1249 KV vaccine 1:1711 MLV/KV vaccine 1 :2044 These results show a 64 % increase with the combined MLV/KV vaccine over the MLV vaccine alone, and a 19% increase over the KV vaccine alone. Of particular interest, the percentage of animals which did not respond, i.e., which showed no increase in titer alter vaccination, was 27% for the KV vaccine and 33% for the MLV vaccine. However, only 15 % animals receiving the combined MLV/KV vaccine of the present invention failed to respond to the vaccinations. Failure of young calves to respond to vaccination (especially BVD) is considered common in the field. EXAMPLE 7 Bovine Parainfluenza Virus - Serotype in (PI-3) A group of 143 young holstein calves (1-4 months old) was divided into three groups and vaccinated with a killed PI-3 vaccine ("KV", Sanofi Animal Health, Inc. List No. 9105), a modified-live form ("MLV", Sanofi List No. 2639), or combined MLV/KV PI-3 vaccine prepared by reconstituting the MLV vaccine, using the KV formulation as diluent. Animals were only given a single dose of each vaccine, intramuscularly. Pre- vaccination and post- vaccination sera were taken, with the post- vaccination sera being collected 35 days after vaccination. These sera were then assayed by a standard serum neutralization test. Mean increases in PI-3 titers were as follows: The data show that the combined MLV/KV vaccine of the present invention resulted in a 110% titer increase over the results obtained with the KV vaccine alone, and an 844 % increase over the results of the MLV vaccine alone. The experimental results set forth in Examples 1-7 for IBR, BVD and PI-3 vaccines all clearly show a marked increase in antibody titers as compared to vaccines that contain only modified-live or killed forms of each virus. As antibody titers often correspond to overall immune status of the animal, the titer serves as an effective measure of immunity that has been generated to an antigen. In contrast to what may be expected, the above results clearly demonstrate a pronounced increase in antibody titers when using the preferred vaccine compositions of the invention. Vaccines of the invention consistently showed increased antibody titers of more than twice, and often more than three or four times, that obtained with either modified-live or killed virus forms alone. In the experiment conducted with PI-3, the vaccine of the invention showed a mean increase in antibody titers of between 9 and 10 times that of the modified-live virus vaccine alone. Similarly, as shown above with IBR, the results obtained with the combined virus of the invention resulted in more than double the antibody titer that was obtained by simply using the modified-live virus vaccine and another, separate dose of the killed virus vaccine. Finally, the results above indicate that the vaccine of the invention is effective for a higher percentage of the animals being tested than either the MLV or KV fractions alone. For example, in Example 4 of the IBR vaccine tests and the test for BVD vaccine, the percentage of test animals that did not significantly respond to vaccination was cut nearly in half, or lower, when using a vaccine of the invention as compared to either the MLV fraction or the KV fraction alone. This finding is particularly significant in very young animals where vaccines often are ineffective in generating an immune system response because of maternal antibodies and the lack of maturity of the immune system EXAMPLE 8 Assessment of Cell-Mediated Responses In order to further understand the improvement demonstrated using combined vaccines of the present invention, experiments were performed to determine whether selected bovine viral vaccines induced virus-specific lymphocyte proliferative responses, leukocyte subset profile changes, virus specific delayed-type hypersensitivity responses, virus-neutralizing antibodies and resistance to IBR challenge. Animals and Experimental Design. Twenty calves susceptible to IBR and BRSV were allotted to five treatment groups of four calves each: Group A = IBR (MLV, Sanofi Animal Health) Group B = IBR (MLV, Smithkline Beecham) Group C = IBR-BRSV (KV, Sanofi Animal Health) Group D = IBR-BRSV (MLV/KV Sanofi Animal Health) Group E = Non-vaccinated controls One week after arrival, calves were vaccinated according to label directions (day 0). Blood samples were obtained from all animals on days 0, 30, 7, 14, 18, 21, and 28. In addition nasal swabs were taken daily 2-10 days following challenge with IBR virus. Lymphocyte Blastogenesis and Subset Analysis. Lymphocytes were isolated and mitogen and antigen-specific lymphocyte proliferative responses and the percentage of cells positive for T helper ("Th"), T cytotoxic ("Tctl") and B lymphocytes were determined. Lymphocytes were incubated with phytohemagglutmin, inactivated IBR, and inactivated BRSV. Control non-stimulated lymphocytes from each calf were incubated only with medium. The percentage of cells positive for CD4, CD8 and immunoglobulin IgM were determined using standard flow microflurometric analysis. Cells were incubated with primary antibody to CD4 (for Th cells), CD8 (for Tctl cells) and IgM (for B cells). Control samples from each calf were treated with phosphate-buffered saline (PBS prior to incubation with the secondary antibody. Cells were washed in PBS, suspended in paraformaldehyde, stored overnight then analyzed on a FACscan (Becton Dickinson) flow cytometer. Delayed-type Hypersensitivity. On day 26, calves were evaluated for delayed-type hypersensitivity (DTH) responses to IBR and BRSV. Injection sites for the DTH responses were located on each side of the neck. Pre-injection skin-fold thickness measurements were taken and calves were then injected intradermally with each viral antigen and PBS. Skin-fold thickness was measured 42 hours later. Skin-fold measurements from PBS injection sites were subtracted form the viral injected skin-fold measurements and then 0-hour measurements were subtracted from the 42-hour measurements. IBR Challenge and Clinical Signs. On day 28, calves were challenged with IBR. Rectal temperatures, clinical signs of respiratory disease, and virus excretion in nasal secretions were monitored for 10 days. Clinical signs were scored as 0 (normal), 1 (mild) or 2 (severe) according to the degree of clinically apparent disease (nasal/ocular discharge and overall appearance). Nasal swab samples were collected daily two days after challenge and submitted for standard virus isolation procedures. Virus Neutralizing Antibody Titers. Serum samples were obtained on each sampling day and used in standard virus neutralization (VN) assays. Results are provided in Tables 1 and 2 below. Lymphocyte Blastogenesis. As expected, lymphocytes isolated from calves in all treatment groups proliferated in response to phytohemagglutmin. IBR-specific lymphocyte proliferative responses are shown in Table 1 below and indicate that responses were highest in the group receiving the MLV/KV vaccine of the invention. Table 1. IBR-specific lymphocyte blastogenesis Values are means of log 10 stimulated counts per minute ("CPM") minus background CPM. Lymphocyte proliferative responses to BRSV were apparent in vaccinated animals (Table 2 below), but responses were assessed only on day 28. Significant treatment differences were not found in the percentages of Th and Tctl lymphocytes during the study. Table 2. BRSM - specific lymphocyte blastogenesis (day 28) Values are means of log I0 stimulated CPM minus background CPM. Delayed-type Hypersensitivity. DTH responses to inactivated IBR and BRSV are shown in Table 3. These data provide convincing evidence that calves receiving killed vaccine virus (Groups C and D) developed cellular immune responses to both IBR and BRSV. Furthermore, the calves receiving the MLV/KV combination IBR vaccine (Group D) showed markedly higher responses than either MLV or KV alone. Table 3. Virus-specific DTH responses Calves were injected intradermally with the antigens and PBS on day 26, 0 hour. Skin-fold thickness (mm) was measured prior to injection (0 hour) and 42 hours later. Saline measurements were subtracted from the antigen measurement; thus, values are means of 42-hour measurements minus 0-hour measurements. IBR Challenge and Clinical Signs. Calves were challenged with IBR (10 8 PFU per calf). In general, none of the calves displayed sever signs of respiratory disease; however, control animals had the highest (most sever) clinical scores. Importantly, on days 4 through 10 post-challenge, calves in Group D had markedly lower clinical scores when compared to Group E (Table 4). Furthermore, less clinical disease was observed in Group D compared to the other vaccinated groups. Table 4. Pooled clinical signs of calves challenged with IBR Values are pooled data of normal (0), mild (1), or severe (2) clinical signs; 5 parameters were considered. IBR Viral Clearance. Virus isolation (VI) results are shown in Table 5. There was a dramatic difference between vaccinated and non-vaccinated animals. The vaccinated calves shed detectable virus fewer total days than ten non-vaccinated calves, i.e. 14 days for group A, 14 days for group B, 13 days for group C, 12 days for group D and 25 days for group E; however, no significant differences were apparent between the vaccinated groups. Table 5. Virology Isolation Results Serology. Virus neutralizing antibody titers are shown in Table 6. The Geometric Mean Titers (GMT) show marked differences between group D and the other groups. It is clear that the animals vaccinated with the combination MLV/KV vaccine of the invention responded to IBR earlier (day 3 vs days 14 or 18) and at a much higher level on each bleeding day post- vaccination. Table 6. IBR Serologic Titers* *Titers expressed as the Geometric Mean Titer (GMT) for each treatment group. Leukocyte Subset Profiles. No differences in leukocyte populations were observed either among the treatment groups or among the days within each group. The key objective of this study was to determine whether there were CMI-related differences between groups of calves vaccinated with vaccines containing modified-live IBR, killed IBR or a modified- live/killed IBR combination. Lymphocyte blastogenesis and DTH data demonstrate the greatest differences between the combination vaccine compared to either the modified-live or killed vaccines alone. These results are consistent with clinical scores where the least evidence of disease was observed in the group vaccinated with the combination vaccine of this invention. Although evaluation of leukocyte subset populations and virus isolation data did not show differences among the vaccinated group, it must be noted that the virus shed after challenge could not be quantified. A second objective was to determine whether IBR-specific humoral immunity (antibodies) showed differences among the groups of calves. The results indicate that the combination vaccine of the invention positively impacted the kinetics of antibody _ production (10-15 days earlier) and the level of antibodies produced (maximums titers ' 5 were 49 versus 20 and 28). A final objective was to determine whether a killed BRSV vaccine (included in the combination IBR vaccine) stimulated a CMI-related response in vaccinated animals. BRSV specific lymphocyte blastogenesis and DTT1 both showed very positive results, i.e., calves receiving the killed BRSV (groups C & D) had values higher than calves not 10 receiving the BRSV vaccine (A & B). The combination IBR vaccine of the invention therefore stimulated higher CMI- related responses and antibodies than either MLV or KV vaccine alone. Although virus shedding data did not differentiate the groups, clinical scores post-challenge favor the IBR combination vaccine group and are consistent with these selected immune parameters. 15 The killed BRSV vaccine stimulated CMI (DTTJ and lymphocyte proliferation) as well. While a preferred embodiment of the present invention has been described, it should be understood that various changes, adaptations and modifications may be made therein without departing from the spirit of the invention and the scope of the appended claims. 20

Claims: CLAIMS: What is claimed is: 1. A method of vaccinating an animal against an infectious agent comprising the step of administering to the animal a vaccine comprising an effective combination of both killed and modified-live forms of the same infectious agent. 2. A method according to claim 1 wherein the vaccine comprises a single, stable formulation in which both forms are provided together in a vehicle suitable for administration to an animal. 3. A method according to claim 1 wherein the vaccine is comprised of a kit, the kit comprising separate formulations of killed and modified-live forms, both formulations being suitable for concurrent administration to an animal. 4. A method according to claim 1 wherein the animal is of the bovine species. 5. A method according to claim 1 wherein the infectious agent is infectious bovine rhinotracheitis virus. 6. A method according to claim 1 wherein the infectious agent is bovine virus diarrhea virus. 7. A method according to claim 1 wherein the infectious agent is bovine respiratory syncytial virus. 8. A method according to claim 1 wherein the infectious agent is parainfluenza-3 virus. 9. A method according to claim 1 wherein the modified-live form of the agent comprises between about 0.1 % and about 1.0% of the total forms of agent present in the vaccine. 10. A vaccine useful for vaccinating an animal against an infectious agent, the vaccine comprising an effective combination of both killed and modified-live forms of the agent. 11. A vaccine according to claim 10 wherein the vaccine comprises a single, stable formulation, in which both forms are provided together in a vehicle suitable for administration to an animal. 12. A vaccine according to claim 10 wherein the animal is of the bovine species. 13. A vaccine according to claim 10 wherein the infectious agent is infectious bovine rhinotracheitis virus. 14. A vaccine according to claim 10 wherein the infectious agent is bovine virus diarrhea. 15. A vaccine according to claim 10 wherein the infectious agent is bovine respiratory syncytial virus. 16. A vaccine according to claim 10 wherein the infectious agent is parainfluenza-3 virus. 17. A vaccine according to claim 7 wherein the modified-live form comprises between about 0.1 % and about 1.0% of the total forms of agent present in the vaccine. 18. A vaccine kit comprising separate formulations of both killed and modified- live forms of an infectious agent, both formulations being suitable for concurrent administration to an animal.

Inventors: ANDERSON GARY A, FIELD MARVIN F, TUCKER CASSIUS M, ANDERSON, GARY, A., FIELD, MARVIN, F., TUCKER, CASSIUS, M.
Applicants: SANOFI ANIMAL HEALTH INC, SANOFI ANIMAL HEALTH, INC.
Application year: 1993
Main classification: A61K39
IPC codes: A61K39