HK1168032B - Enhanced immune response in avian species - Google Patents

Description

Enhanced immune response in birds

The present invention relates to a method of immune activation in birds. In particular, the invention includes methods for eliciting respiratory and systemic, non-specific, and antigen-specific immune responses that are useful for immunizing, vaccinating, and treating animals against infectious diseases.

Vaccines are used to prevent infectious diseases and to treat established diseases. Infectious diseases are caused by infectious agents (including such examples as viruses, bacteria, parasites, and other fungi). Numerous agents and methods have been developed for the prevention and treatment of infectious diseases for all types of species, including mammals, birds, fish and primates.

The vaccination procedure is particularly important for commercially farmed animals used in the food industry. Birds are a major target for many types of infection. Breeders and commercial chicken, turkey, and other poultry populations are often inoculated to protect them against environmental exposure to pathogens. One of the most economically important diseases for the poultry industry is Marek's disease, a naturally occurring lymphoproliferative disease in chickens. The disease is caused by highly contagious herpesviruses which spread horizontally and cause major economic losses in the poultry industry. The symptoms of Marek's disease are widespread in nerves, reproductive organs, internal organs, eyes and skin of infected birds, causing motor paralysis, impaired organ function and chronic malnutrition, ultimately leading to death.

Hatched birds are exposed to pathogenic microorganisms shortly after birth. Although these birds are initially protected against pathogens by maternal derived antibodies, this protection is only temporary and the birds' own immature immune system must begin to protect the birds against pathogens. It is often desirable to prevent infection in hatchlings, when they are most susceptible to infection. It is also desirable to prevent infection in older birds, especially when the birds are housed in close quarters, leading to rapid spread of the disease.

In most commercial poultry farms certain vaccines are administered parenterally to newly hatched chicks at hatching. Because exposure to pathogens often occurs at a very young age, they often need to be inoculated before they are placed in a rearing or incubation room. Such vaccination protocols require the handling of a single bird, involving the possible risk of accidental autoinjection. Furthermore, these vaccines are not always effective. Young chicks may be exposed to toxic forms of the disease shortly after vaccination before they have had the opportunity to develop adequate protective immunity.

Some live virus vaccines can be administered in eggs before the birds hatch. This method is called "in ovo inoculation". Birds that are inoculated in ovo develop resistance to the target disease. However, many vaccines used to hatch birds cannot be used for in ovo vaccination because of the embryopathogenicity of the vaccine agent. Late embryos are highly sensitive to infection by most of the vaccine viruses tested. Not all vaccine viruses that are not pathogenic to newly hatched chicks are also safe for late-stage embryos. For example, vaccine strains of Infectious Bronchitis Virus (IBV) and Newcastle Disease Virus (NDV), which are commonly used as a newborn vaccine for newly hatched chicks, are lethal to embryos after in ovo inoculation. These viruses have been engineered to render them safe for in ovo use. The alteration of the virus attenuates the immune response elicited and is therefore a vaccine agent with reduced efficacy in protecting late-stage embryos.

Vaccination procedures resulting from such different vaccines that need to provide protection against infectious diseases and economic losses are complex. Thus, there is a need for a method of eliciting a non-antigen specific immune response that enhances the protection of birds from infectious diseases and that is easy to administer. Furthermore, it would be desirable to have a method of eliciting an immune response that provides protective function against more than one infectious agent. There is also a need for a method of eliciting an immune response that has a longer duration or does not require booster injections of a vaccine. The present invention provides a method of eliciting an avian non-antigen specific immune response that also reduces the incidence of populations, provides a protective function against more than one infectious agent, and provides protection for a longer period of time than other products commonly known in the art.

Brief Description of Drawings

Figure 1 is a graph illustrating hatchability between different treatment groups of embryonated eggs. The study groups analyzed included T1, 0 micrograms immunomodulator/egg and E.coli challenge, T2, 0.1 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T4,10.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and no challenge, and T6, 1.0 micrograms immunomodulator/egg and no challenge.

Figure 2 illustrates the average daily mortality per chicken house per day after hatching. Diagram (key): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 3 is a graph showing the proportion of bird survival per chicken house on any particular study day based on the initial number of embryonated eggs. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

FIG. 4 graphically illustrates the viability of birds after hatch. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 5 illustrates survival, hatch mortality and post-hatch mortality during the 7 day study. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

FIG. 6 graphically depicts survival, hatch mortality and post hatch mortality by study day 14. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

Figure 7 graphically depicts survival, hatch mortality and post hatch mortality by study day 21. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

Figure 8 graphically illustrates survival, hatch mortality and post hatch mortality by study day 28. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

FIG. 9 graphically depicts survival, hatch mortality, and post hatch mortality by study day 35. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

FIG. 10 graphically depicts survival, hatch mortality, and post hatch mortality by study day 45. Scheme (b): t1, 0 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T5, 0 micrograms immunomodulator/egg and non-challenge, and T6, 1.0 micrograms immunomodulator/egg and non-challenge.

Figure 11 shows the cumulative mortality rate for each group from 18-day-old embryos by study day 45. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 12 shows the cumulative mortality of each group after incubation from study day 0 to study day 45. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 13 shows the total average body weight gain observed from study day 0 to day 7. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 14 shows the total average body weight gain observed from study day 0 to day 14. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 15 shows the total average body weight gain observed from study day 0 to day 21. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 16 shows the total average body weight gain observed from study day 0 to day 28. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 17 shows the total average body weight gain observed from study day 0 to day 35. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

Figure 18 shows the total mean body weight gain observed from study day 0 to day 45. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,10.0 microgram immunomodulator/egg and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and non-challenge, and T6, 1.0 microgram immunomodulator/egg and non-challenge.

FIG. 19 graphically depicts the number of chickens hatched per study group of embryonated chicken eggs. The study groups analyzed included T1, 0 micrograms immunomodulator/egg and E.coli challenge, T2, 0.1 micrograms immunomodulator/egg and E.coli challenge, T3,1.0 micrograms immunomodulator/egg and E.coli challenge, T4,1.0 micrograms immunomodulator/egg plus 1 dose of Marek's vaccine and E.coli challenge, T5, 0 micrograms immunomodulator/egg and no challenge, and T6, 1.0 micrograms immunomodulator/egg and no challenge.

Figure 20 is a graph showing the number of live chickens per group on study day 7. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,1.0 microgram immunomodulator/egg plus 1 dose of Marek's vaccine and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and no challenge, and T6, 1.0 microgram immunomodulator/egg and no challenge.

Figure 21 shows a comparison of live and dead chickens per group on study day 7.

Figure 22 shows the percent mortality for each study group by study day 7. Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,1.0 microgram immunomodulator/egg plus 1 dose of Marek's vaccine and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and no challenge, and T6, 1.0 microgram immunomodulator/egg and no challenge.

Figure 23 illustrates the number of surviving birds at the end of the study (day 7) for each group including dead embryos, dead chickens and live chickens (green). Scheme (b): t1, 0 microgram immunomodulator/egg and escherichia coli challenge, T2, 0.1 microgram immunomodulator/egg and escherichia coli challenge, T3,1.0 microgram immunomodulator/egg and escherichia coli challenge, T4,1.0 microgram immunomodulator/egg plus 1 dose of Marek's vaccine and escherichia coli challenge, T5, 0 microgram immunomodulator/egg and no challenge, and T6, 1.0 microgram immunomodulator/egg and no challenge.

FIG. 24 graphically depicts the percentage of chicks hatched per study group of embryonated eggs. The study groups analyzed included those listed in table 4.

FIG. 25 shows the percent mortality after incubation for the study groups not challenged with E.coli by study day 7. The study groups analyzed included those listed in table 4.

FIG. 26 shows the percent mortality of the study groups challenged with E.coli from post-hatch to study day 7. The study groups analyzed included those listed in table 4.

Figure 27 graphically depicts the percent mortality of each study group from post-hatch to study day 7. The study groups analyzed included those listed in table 4.

Figure 28 shows the percent survival of each study group after incubation until study day 7. The study groups analyzed included those listed in table 4.

Figure 29 shows the percent survival of each study group on study day 7. The study groups analyzed included those listed in table 4.

Figure 30 shows the weekly mortality of each study group by study day 7 and 14. The study groups analyzed included a subset of those listed in table 4.

Figure 31 graphically depicts the effect of immunomodulators on MD vaccine replication of HVT in splenocytes in the first week of the study. The study groups analyzed included those listed in table 5.

Figure 32 graphically depicts the effect of immunomodulators on MD vaccine replication of HVT in PMBC in the first week of the study. The study groups analyzed included those listed in table 5.

FIG. 33 graphically depicts the effect of immunomodulators on MD vaccine replication of SB-1 in splenocytes in the first week of the study. The study groups analyzed included those listed in table 5.

FIG. 34 graphically depicts the effect of immunomodulators on MD vaccine replication of SB-1 in PMBC in the first week of the study. The study groups analyzed included those listed in table 5.

Figure 35 graphically depicts the effect of immunomodulators on MD vaccine replication of HVT in splenocytes in a second week of study. The study groups analyzed included those listed in table 5.

Figure 36 graphically depicts the effect of immunomodulators on MD vaccine replication of HVT in PMBC in the second week of the study. The study groups analyzed included those listed in table 5.

FIG. 37 graphically depicts the effect of immunomodulators on MD vaccine replication of SB-1 in splenocytes in a second week of study. The study groups analyzed included those listed in table 5.

FIG. 38 graphically depicts the effect of immunomodulators on MD vaccine replication of SB-1 in PMBC in the second week of the study. The study groups analyzed included those listed in table 5.

Figure 39 graphically depicts the reduction of aerocystitis in ovo immune modulators, anti-ND vaccination and challenged chicks. The study groups analyzed included those listed in table 7.

FIG. 40 graphically depicts ELISAs of the study groups listed in Table 7 on day 26.

FIG. 41 graphically depicts survival rates of chicks in the study groups listed in Table 8.

FIG. 42 graphically depicts the incidence of Marek's disease in the study group listed in Table 8.

Detailed Description

The method of eliciting an immune response in an avian member of the present invention comprises administering to the avian member an effective amount of an immunomodulator composition to elicit an immune response in the avian member. The immunomodulator composition comprises a liposome delivery vehicle and at least one nucleic acid molecule. In particular, the immunomodulator elicits a non-antigen specific immune response that is further enhanced by administration of at least one biological agent, such as a vaccine.

The methods provide novel therapeutic strategies for protecting birds from infectious diseases and for treating populations with infectious diseases. Furthermore, by using the immunomodulator composition of the present invention, there is no decrease in hatchability or post-hatch survival when the immunomodulator is administered in ovo, nor when co-administered with a vaccine. In addition, pre-administration of an immunomodulator prior to administration of the vaccine further enhances a non-antigen specific immune response. The method of the invention also allows vaccines that have previously only been administered post-hatch to be safely used in ovo. Furthermore, the methods of the invention allow for the combination of more than one vaccine with an immunomodulator composition. Finally, when an immunomodulator is used in combination with a vaccine, the methods of the present invention provide longer protection against disease.

I. Composition comprising a metal oxide and a metal oxide

a. Immunomodulator

In one embodiment of the invention, an immunomodulator composition comprises a liposome delivery vehicle and at least one nucleic acid molecule, as described in U.S. patent No. 6,693,086, which is incorporated herein by reference.

Suitable liposome delivery vehicles include lipid compositions capable of delivering nucleic acid molecules to the tissue of the subject being treated. The liposome delivery vehicle is preferably stable for a sufficient amount of time in the subject to deliver the nucleic acid molecule and/or biological agent. In one embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 30 minutes. In another embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 1 hour. In yet another embodiment, the liposome delivery vehicle is stable in the recipient subject for at least about 24 hours.

The liposome delivery vehicles of the present invention include lipid compositions that are capable of fusing with the plasma membrane of a cell to deliver a nucleic acid molecule into the cell. In one embodiment, when the nucleic acid: liposome complex of the invention is delivered, at least about 1 picogram (pg) of protein per milligram (mg) of total tissue protein per microgram (μ g) of nucleic acid delivered is expressed. In another embodiment, the transfection efficiency of the nucleic acid: liposome complex is at least about 10 pg protein per mg total tissue protein per microgram of delivered nucleic acid expressed; and in yet another embodiment, at least about 50 pg protein is expressed per mg total tissue protein per microgram of nucleic acid delivered. The transfection efficiency of the complex may be as low as 1 nanogram (fg) of protein per mg of total tissue protein per microgram of nucleic acid delivered, with the above amounts being more preferred.

Preferred liposomal delivery vehicles of the invention are those having a diameter of about 100 to 500 nanometers (nm), in another embodiment, a diameter of about 150 to 450 nm and in yet another embodiment, a diameter of about 200 to 400 nm.

Suitable liposomes include any liposome, such as those commonly used in, for example, gene delivery methods known to those skilled in the art. Preferred liposome delivery vehicles include multilamellar vesicle (MLV) lipids and extruded lipids. Methods for preparing MLV's are well known in the art. More preferred liposome delivery vehicles include liposomes having a polycationic lipid composition (i.e., cationic liposomes) and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Exemplary cationic liposome compositions include, but are not limited to, N- [1- (2, 3-dioleyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTMA) and cholesterol, N- [1- (2, 3-dioleoyloxy) propyl]-N, N, N-trimethylammonium chloride (DOTAP) and cholesterol, 1- [2- (oleoyloxy) ethyl]-2-oleyl-3- (2-hydroxyethyl) imidazolineChloride (DOTIM) and cholesterol, dimethyldioctadecylammonium bromide (DDAB) and cholesterol, and combinations thereof. The most preferred liposome composition for use as a delivery vehicle includes DOTIM and cholesterol.

Suitable nucleic acid molecules include any nucleic acid sequence, such as coding or non-coding sequences, as well as DNA or RNA. The coding nucleic acid sequence encodes at least a portion of a protein or peptide, while the non-coding sequence does not encode any portion of a protein or peptide. According to the invention, a "non-coding" nucleic acid may comprise a regulatory region of a transcription unit, such as a promoter region. The term "empty vector" is used interchangeably with the term "non-coding", and particularly refers to a nucleic acid sequence in which no protein-coding portion is present, such as a plasmid vector that does not contain a gene insert. Expression of the protein encoded by the nucleic acid molecule is not required to elicit a non-antigen specific immune response; thus, the nucleic acid molecule need not be operably linked to a transcriptional regulatory sequence. However, further advantages (i.e., antigen specificity and enhanced immunity) can be obtained by including nucleic acid sequences (DNA or RNA) encoding the immunogen and/or cytokine in the composition.

Complexation of liposomes with nucleic acid molecules can be achieved using methods standard in the art or described in U.S. patent No. 6,693,086 (incorporated herein by reference). Suitable concentrations of nucleic acid molecules added to the liposomes include those effective to deliver sufficient amounts of nucleic acid molecules into a subject such that a systemic immune response is elicited. When the nucleic acid molecule encodes an immunogen or a cytokine, suitable concentrations of the nucleic acid molecule added to the liposome include concentrations effective to deliver a sufficient amount of the nucleic acid molecule into the cell so that the cell can produce sufficient immunogen and/or cytokine protein to modulate effector cell immunity in a desired manner. In one embodiment, about 0.1 micrograms to about 10 micrograms of nucleic acid molecules are combined with about 8 nmol liposomes, in another embodiment, about 0.5 micrograms to about 5 micrograms of nucleic acid molecules are combined with about 8 nmol liposomes, and in yet another embodiment, about 1.0 micrograms of nucleic acid molecules are combined with about 8 nmol liposomes. In one embodiment, the ratio of nucleic acids to lipids in the composition (micrograms nucleic acids: nmol lipids) is at least about 1:1 nucleic acids: lipids (i.e., 1 micrograms nucleic acids: 1 nmol lipids), and in another embodiment, at least about 1:5, and in yet another embodiment, at least about 1:10, and in a further embodiment, at least about 1:20, by weight. The ratios expressed herein are based on the amount of cationic lipid in the composition, and not on the total amount of lipid in the composition. In another embodiment, the ratio of nucleic acid to lipid in the compositions of the invention is from about 1:1 to about 1:64 nucleic acid to lipid by weight; and from about 1:5 to about 1:50 nucleic acid: lipid, by weight, in another embodiment; and in further embodiments, from about 1:10 to about 1:40 nucleic acid: lipid, by weight; and from about 1:15 to about 1:30 nucleic acid: lipid, by weight, in yet another embodiment. Another ratio of nucleic acid to lipid is about 1:8 to 1:16, with about 1:8 to 1:32 being preferred.

b. Biological agent

In another embodiment of the invention, the immunomodulator comprises a liposome delivery vehicle, a nucleic acid molecule, and at least one biologic.

Suitable biological agents are agents effective in preventing or treating avian diseases. Such biologies include immunopotentiating proteins, immunogens, vaccines, or any combination thereof. Suitable immune enhancing proteins are those proteins known to enhance immunity. By way of non-limiting example, cytokines (including protein families) are a family of proteins known to enhance immunity. Suitable immunogens are proteins that elicit a humoral and/or cellular immune response such that administration of the immunogen to a subject achieves an immunogen-specific immune response against the same or similar proteins encountered within the tissues of the subject. Immunogens may include pathogenic antigens expressed by bacteria, viruses, parasites, or fungi. Preferred antigens include antigens that cause infectious disease in a subject. According to the present invention, an immunogen may be any part of a protein of natural or synthetic origin, which elicits a humoral and/or cellular immune response. Thus, the size of an antigen or immunogen may be as small as about 5-12 amino acids, as large as the full length protein, including sizes in between. The antigen may be a multimeric protein or a fusion protein. The antigen may be a purified peptide antigen derived from natural or recombinant cells. The nucleic acid sequences of the immunopotentiating protein and the immunogen are operably linked to transcriptional regulatory sequences such that the immunogen is expressed in the tissue of the subject, thereby eliciting an immunogen-specific immune response (in addition to a non-specific immune response) in the subject.

In another embodiment of the invention, the biological agent is a vaccine. The vaccine may comprise a live, infectious virus vaccine or an inactivated, inactivated virus vaccine. In one embodiment, one or more vaccines, live or inactivated viral vaccines, may be used in combination with the immunomodulator composition of the present invention. Suitable vaccines include those known in the art for use in birds. Exemplary vaccines, without limitation, include those used in the art to protect against: marek's Disease Virus (MDV), Newcastle Disease Virus (NDV), Chicken Anemia Virus (CAV), Infectious Bursal Disease Virus (IBDV), Infectious Bronchitis Virus (IBV), Herpes Virus of Turkeys (HVT), infectious laryngotracheitis virus (ILTV), Avian Encephalomyelitis Virus (AEV), fowlpox virus (FPV), fowl cholera, Avian Influenza Virus (AIV), reovirus, Avian Leukosis Virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus and Hemorrhagic Enteritis Virus (HEV), coccidia, and other diseases known in the art. In another example, the vaccine can be the vaccine described in U.S. Pat. nos. 5,427,791, 6,048,535 and 6,406,702. In an exemplary embodiment, a vaccine for the protection of Marek's disease may be used in combination with an immunomodulator composition of the present invention.

Process II

a. Method of immunostimulation

In one embodiment of the invention, an immune response is elicited in an avian member by administering to the avian member an effective amount of an immunomodulator composition. The effective amount is sufficient to elicit an immune response in the avian member. Immunomodulators include liposome delivery vehicles and nucleic acid molecules.

In one embodiment, an effective amount of an immunomodulator is from about 0.05 micrograms to about 10 micrograms. In another embodiment, the effective amount of an immunomodulator is from about 0.1 micrograms to about 5 micrograms.

In another embodiment of the invention, an immune response is elicited in a member of the bird by administering an effective amount of an immunomodulator composition (including liposomal delivery vehicles, isolated nucleic acid molecules and biologics). It is contemplated that the biological agent may be co-administered with the immunomodulator or administered independently thereof. The single administration may be in the absence of administrationBefore or after the regulator. It is also contemplated that more than one administration of an immunomodulator or biological agent can be used to prolong enhanced immunity. In addition, more than one biological agent may be co-administered with an immunomodulator, administered prior to an immunomodulator, or administered after an immunomodulator, as described in the examples herein. In one embodiment, an effective amount of an immunomodulatory agent is about 0.05 micrograms to about 5 micrograms of a liposome delivery vehicle and isolated nucleic acid molecule and about 300 to about 30000 TCID₅₀A viral biological agent. In another embodiment, an effective amount of an immunomodulatory agent is about 0.1 micrograms to about 3 micrograms of a liposome delivery vehicle and isolated nucleic acid molecule and about 100 to about 1000 EID₅₀A viral biological agent.

b. Disease and disorder

The methods of the invention elicit an immune response in a subject, such that the subject is protected from a disease that will elicit an immune response. As used herein, the phrase "protect against disease" refers to a reduction in disease symptoms; a reduction in the incidence of disease; and reduction in disease severity. Protecting a subject may refer to the ability of a therapeutic composition of the invention, when administered to a subject, to prevent the appearance of a disease and/or to cure or alleviate the symptoms, signs or causes of a disease. Thus, protection of avian members from disease includes prevention of disease development (prophylactic treatment) and treatment of avian members with disease (therapeutic treatment). Specifically, protection of a subject from disease is achieved by: an immune response is elicited in an avian member by inducing a beneficial or protective immune response, which may in some cases additionally suppress, mitigate, inhibit or block an excessive or harmful immune response. The term "disease" refers to any deviation from the normal health status of an avian member, including the following states: when disease symptoms are present, and deviations (e.g., infection, genetic mutation, genetic defect, etc.) from conditions that already exist but for which symptoms have not yet been shown.

The methods of the invention can be used to prevent disease, stimulate effector cell immunity against disease, clear disease, alleviate disease, and prevent secondary disease arising from the primary disease.

The methods of the invention comprise administering a composition to protect against infection by a variety of pathogens. The composition administered may or may not include a specific antigen that elicits a specific response. It is contemplated that the methods of the invention will protect recipient subjects from diseases derived from infectious microbial sources, including, but not limited to, viruses, bacteria, fungi, and parasites. Exemplary viral infectious diseases, without limitation, include those resulting from viral infections: chicken Infectious Anemia Virus (CIAV), Marek's Disease Virus (MDV), chicken herpes virus (HCV), turkey herpes virus (HTV), Infectious Bursal Disease Virus (IBDV), Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), infectious laryngotracheitis virus (ILTV), paramyxovirus type 3, Avian Encephalomyelitis (AEV), fowlpox virus (FPV), fowl cholera, Avian Influenza Virus (AIV), reovirus, Avian Leukosis Virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus, Hemorrhagic Enteritis Virus (HEV), pneumovirus, pigeon pox virus, recombinants thereof, and other viruses known in the art. Exemplary bacterial infections, without limitation, include those derived from bacterial infections of: gram-positive or negative bacteria and fungi such as Escherichia coli, Mycoplasma gallisepticum (Mycoplasma gallisepticum), Mycoplasma hominus (Mycoplasma meliagris), Mycoplasma synoviae (Mycoplasma synoviae), Bordetella (Bordetella Sp), Pasteurella multocida (Pasteurella multocida), Clostridium perfringens (Clostridium perfringens), Clostridium enterobacter (Clostridium colinum), Campylobacter jejuni (Campylobacter jejuni), Salmonella S (Salmonella Sp), Salmonella enteritidis (Salmonella enteritidis), Salmonella pullorum (Salmonella pulorum), Salmonella gallinarum (Salmonella gallinarum), Clostridium (Bordetella botuli), Clostridium gallinarum (Salmonella gallinarum), Salmonella gallinarum (Salmonella gallinarum), Rhodococcus bacterium (Salmonella anatis), and other bacteria known in the art (Rhodococcus bacterium), Rhodococcus bacterium (Eriphora), and Salmonella anatipes. Exemplary fungal or mold infections, without limitation, include those resulting from: aspergillus fumigatus (Aspergillus fumigates), Aspergillus flavus (Aspergillus flavus), Candida albicans (Candida albicans), and other infectious fungi or molds known in the art. Exemplary disease conditions, without limitation, include those derived from the following toxins: toxins from gram-positive or gram-negative bacteria and fungi such as clostridium perfringens (clostridium perfringens) toxin, botulinum toxin, escherichia coli enterotoxin, staphylococcus toxin, pasteurella leukotoxin, and fusarium mycotoxin as well as other toxins known in the art. Exemplary parasites include, but are not limited to, Eimeria zeylanica (Eimeria melegaridis), Coccidia (coccidia sp), ascaris galli (Ascaridia galli), Heterorhabdus gallinarum (Heterakis gallinae), Trichinella circinata (Capillaria annulata), Trichinella torsades (Capillaria contorta), Coprinus pigeon (Capillaria obsignata), Heterorhabdus tracheaelis (Syngamus trachela), and other parasites known in the art.

c. Test subject

The methods of the invention may be administered to any subject or member of an avian, whether domesticated or wild. In particular, it can be administered to those subjects in commercial rearing for breeding, meat production or egg production. Suitable avian subjects include, without limitation, chickens, turkeys, geese, ducks, pheasants, quail, pigeons, ostriches, caged birds, zoologically-harvested birds and birdhouses, and the like. In one embodiment, the avian member is selected from a chicken or a turkey. The skilled person will appreciate that the method of the invention will be very beneficial for birds raised in high density hatcheries, such as broiler chickens and egg-laying chickens, as they are particularly vulnerable to environmental exposure to infectious agents.

d. Administration of

Various routes of administration may be provided. The particular manner chosen will, of course, depend on the particular biological agent selected, the age and general health of the subject, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of the invention may be practiced with any mode of administration that produces an effective level of immune response and does not cause clinically unacceptable side effects. The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art.

The inoculation of birds may be performed at any age. Generally, 18-day-old embryos (in ovo) and above are inoculated with live microorganisms, and 3 weeks old and above are inoculated with inactivated microorganisms or other types of vaccines. For in ovo inoculation, inoculation may be performed during the post-1/4 period of embryo development. The vaccine may be administered by: subcutaneous, feather, by spraying, oral, intraocular, intratracheal, nasal, intraegg or by other methods known in the art. Oral vaccines can be administered in drinking water. Further, it is contemplated that the methods of the present invention may be used based on conventional vaccination protocols. In one embodiment, an immunomodulatory composition of the invention is administered in ovo. In another embodiment, the immunomodulator composition is administered as a spray after an e.

Other delivery systems may include timed release, delayed release or slow release delivery systems. Such systems can avoid repeated application of the composition, thereby increasing convenience. Many types of delivery systems are known and known to those of ordinary skill in the art. They include polymer-based systems such as poly (lactide-co-glycolide), copolyoxalates, polycaprolactone, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules comprising the above-described polymers of drugs are described, for example, in U.S. Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, which are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral lipids such as mono-, di-and triglycerides; a hydrogel release system; the sylastic system; a peptide-based system; a wax coating; compressed tablets using conventional binders and excipients; a partially fused implant; and so on. Specific examples include, but are not limited to, eroding systems, wherein the formulations of the present invention are contained in an intramatrix form, such as those described in U.S. Pat. nos. 4,452,775, 4,675,189, and 5,736,152, and dispersion systems, wherein the active ingredient permeates from the multimer at a controlled rate, such as those described in U.S. Pat. nos. 3,854,480, 5,133,974, and 5,407,686. In addition, pump-based hardware delivery systems may be used, some of which are suitable for implantation.

As various changes could be made in the above compositions, products, and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the following examples shall be interpreted as illustrative and not in a limiting sense.

Definition of

The term "effective amount" refers to an amount necessary or sufficient to achieve the desired biological effect. For example, an effective amount of an immunomodulator for use in the treatment or prevention of an infectious disease is that amount which, when exposed to a microorganism, is necessary to elicit the appearance of an antigen-specific immune response, thereby resulting in a reduction in the amount of microorganisms and, preferably, the destruction of the microorganisms in a subject. The effective amount for any particular application may vary depending on such factors as the disease or disorder to be treated, the size of the subject, or the severity of the disease or disorder. Effective amounts of immunomodulators can be determined empirically by one of ordinary skill in the art without undue experimentation.

The term "cytokine" refers to a family of proteins that enhance immunity. The cytokine family includes hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules, and chemokines (i.e., proteins that regulate the migration and activation of cells, particularly phagocytes). Exemplary cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-12 (IL12), interleukin-15 (IL-15), interleukin-18 (IL-18), interferon-alpha (IFN α) interferon- α (IFN α), and interferon- α (IFN α).

The term "trigger" may be used interchangeably with the terms activate, stimulate, produce or upregulate.

The term "eliciting an immune response" in a subject refers to an activity that specifically controls or affects an immune response, and may include activating an immune response, up-regulating an immune response, enhancing an immune response, and/or altering an immune response (such as by eliciting an immune response that in turn changes the type of immune response that predominates in a subject from a deleterious or ineffective type to a beneficial or protective type).

The term "operably linked" refers to the linkage of a nucleic acid molecule to a transcriptional regulatory sequence in a manner that enables expression of the molecule when transfected (i.e., transformed, transduced or transfected) into a host cell. Transcriptional regulatory sequences are sequences that control the initiation, extension, and termination of transcription. Particularly important transcriptional regulatory sequences are those that control initiation of transcription, such as promoters, enhancers, operators, and repression sequences. Various such transcriptional regulatory sequences are known to those skilled in the art. Preferred transcriptional regulatory sequences include those that function in avian, fish, mammalian, bacterial, plant and insect cells. Although any transcriptional regulatory sequence may be used in the present invention, the sequence may include a naturally occurring transcriptional regulatory sequence that is naturally associated with a sequence encoding an immunogen or an immunostimulatory protein.

The terms "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably and include DNA, RNA, or derivatives of DNA or RNA. The term also includes oligonucleotides and larger sequences, including nucleic acid molecules that encode proteins or fragments thereof, as well as nucleic acid molecules that include regulatory regions, introns, or other non-coding DNA or RNA. Typically, the oligonucleotide has a nucleic acid sequence of about 1 to about 500 nucleotides in length, and more typically, at least about 5 nucleotides in length. The nucleic acid molecule may be derived from any source, including mammalian, fish, bacterial, insect, viral, plant, or synthetic sources. Nucleic acid molecules can be produced by methods generally known in the art, such as recombinant DNA techniques (e.g., Polymerase Chain Reaction (PCR), amplification, cloning) or chemical synthesis. Nucleic acid molecules include natural nucleic acid molecules and homologs thereof, including, but not limited to, natural allelic variants and engineered nucleic acid molecules in which nucleotides have been inserted, deleted, substituted or inverted in a manner such that such modifications do not substantially interfere with the ability of the nucleic acid molecule to encode an immunogen or an immunostimulatory protein useful in the methods of the invention. Nucleic acid homologs can be generated using a number of methods known to those of skill in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989), which is incorporated herein by reference. Techniques for screening for immunogenicity (such as pathogen antigen immunogenicity or cytokine activity) are known to those skilled in the art and include various in vitro and in vivo assays.

Examples

The following examples illustrate various embodiments of the present invention.

Example 1. materials and methods.

Immunomodulator

The immunomodulator used in this study was a composition comprising a cationic lipid and non-coding DNA. Formulating a synthetic immunomodulatory lipid component [1- [2- [9- (Z) -octadecenoyloxy [ ] -]]-2-[8](Z) -heptadecenyl]-3- [ hydroxyethyl group]ImidazolineChloride (DOTIM) and the synthetic neutral lipid cholesterol to produce liposomes of approximately 200 nm in diameter (see, U.S. patent 6,693,086). The DNA component is a 4292 base pair non-coding DNA plasmid (pMB 75.6) produced in e.coli, which is negatively charged, combined with positively charged (cationic) liposomes (see, us patent 6,693,086).

Table 1. dose immune modulator dilution protocol administered to 600 eggs/group.

Research animals

Commercial supply eggs (broiler) were checked against light during 18 days of incubation to determine activity. Healthy embryos were included in the study and infertile, dead and unhealthy embryos were discarded. Chicks were placed in a conventional california type chicken house with curtains on the sides. Each chicken house had 50 unidentified birds on day 0. Chicks were fed a diet that met the MRC recommendations for age and weight of the study animals, and were available ad libitum via bell drinkers connected to the water supply of each chicken house.

Experimental infection and challenge

The chicks were challenged with organisms to determine the efficacy of the immune response. Challenge or experimental infection, including exposure to an inoculum of an organism such as e. The use concentration is 2.63 × 10⁵And administered by spraying 0.15 mL of inoculum onto each embryonated egg (in the hatching tray).

Example 2 administration of an immunomodulator increases hatchability.

A study was conducted to determine the effect of the immunomodulator as described in example 1 administered to 18 day old embryonated chicken eggs (which were then exposed to e. The study included two groups (Table 2), one challenged with E.coli (T1-4) and the other not challenged (T5 and T6). In the e.coli challenge group, there were four subgroups, each subgroup administered a different dose of immunomodulator, including the following doses: none (T1), 0.1 microgram (T2), 1.0 microgram (T3), and 10.0 microgram (T4). The non-challenged group included two subgroups, which were administered no immunomodulator (T5) or 1.0 microgram immunomodulator (T6).

Table 2. study treatment group.

On study day 0, in ovo injections of 18-day-old embryonated eggs receiving immunomodulators were performed. On day 1, 0.15 mL of each 19-day-old embryonated egg in the challenge group (T1-T4) was sprayed at a concentration of 2.63X 10⁵An inoculum of Escherichia coli of (1).

Analysis of the entire panel resulted in a statistically significant treatment effect on embryonated egg hatchability. There was no significant difference between the two non-challenged controls (T5, 0 micrograms/kg, 91% versus T6, 1.0 micrograms/kg, 89%). Furthermore, the comparison between the two untreated groups (T1-challenge and T5-non-challenge) resulted in more embryonated eggs in the non-challenged group hatching (85% versus 91%, respectively). Therefore, the challenge model was confirmed as an effective model for evaluating hatchability.

For those groups treated with immunomodulators, the T3 (1.0 μ g/kg-treated eggs) group hatched more birds (91.5%) than either untreated/challenged T1 (84.5%) or T4 (10.0 μ g/kg-treated eggs) (85%) (fig. 1). Similar differences were shown between the untreated/non-challenged control group (T5) and the two previously described treatment groups (T1, and T4, as described above). No other important findings were shown in any of the remaining pairwise (pair-wise) group comparisons (fig. 1).

There was no significant difference in the proportion of bird deaths in each chicken house, group, each study day (fig. 2). The results of the proportion of bird survival on any particular study day were not statistically significant in relation to treatment and time, but showed significant treatment effects (figure 3). Birds from groups T3, T5, and T6 had significantly more birds on any day when compared to groups T1, T2, and T4. Furthermore, T5 birds also have significantly higher activity than T3 birds. Evaluation of post-hatch bird viability produced important findings similar to those described above (fig. 4 to 12).

There was no significant difference in the observed weight of the henhouse on days 7, 14, 21 or 28 (fig. 13 to 16). Analysis of total henhouse weight on day 35 yielded significant differences between the T5 and T2 groups (mean 87 kgs versus 75 kgs, respectively) (fig. 17). At the end of the study (day 45), the henhouse weights were greater for groups T5 (120 kgs) and T6 (117 kgs) when compared to T1 (105 kgs), and T5 was greater when compared to T2 (106 kgs) and T4 (105 kgs) (fig. 18).

In summary, the effect of immunomodulators when administered in ovo (prior to e.coli infection) in commercial broilers was evaluated. For those groups treated with immunomodulators, the T3 group (92%) hatched more birds than the untreated/challenged T1 (85%) or T4 (85%). Similar differences were shown between the untreated/non-challenged control group (T5) and the same treated groups T1 and T4. There were no significant differences in mortality (fig. 2 to 12) and body weight (fig. 13 to 18). In this study, the immunomodulator was effective in increasing the hatchability of commercial broiler eggs (challenged with E.coli before hatch), and in particular a dose of 1.0 μ g indicated enhanced immunity in the immunomodulator-receiving group.

Example 3 administration of immunomodulators increases non-antigen specific immunity.

A study was conducted to determine the effect of the immunomodulator as described in example 1 in Marek's disease vaccinated chickens (exposed to e. The study included two groups, one challenged with E.coli (T1-T4) and the other not (T5 and T6). In the e.coli challenge group, there were four subgroups, each subgroup administered a different dose of immunomodulator, including the following doses: none (T1), 0.1 microgram (T2), 1.0 microgram (T3), 1.0 microgram plus 1 dose of Marek's vaccine (T4) (table 3). The non-challenged group included two subgroups, which were administered no immunomodulator (T5) or 1.0 microgram immunomodulator (T6).

Table 3. study treatment group.

Incubating the commercial broiler eggs for 18 days, examining viability against light, and then subjecting the 18-day-old chickens receiving the immunomodulator toThe embryonated eggs are injected in ovo. The next day, 0.15 mL of each 19-day-old embryonated egg in the challenge group was sprayed to a concentration of 2.8X 10⁷Inoculum of E.coli per ml. Eggs hatch on study day 0, which extends for 45 days.

The number of embryonated eggs hatched per group (n =1000 per group or hatching tray) was recorded (fig. 19). Among the hatched chicks, the number of chicks still alive at day 7 after hatching was recorded (fig. 20). The number of live/dead chickens per group at day 7 after hatching was also recorded (fig. 21). The percent mortality in the challenge group decreased with increasing immunomodulators, and co-administration of Marek's vaccine with immunomodulators further decreased the percent mortality (figure 22).

The data show that there was no reduction in hatchability between the immunomodulator-treated and untreated groups (not challenged with e.coli) (figure 23). Hatchability was significantly higher between the immunomodulator-treated and untreated groups (challenged with e. The hatchability of embryonated eggs receiving immunomodulators and a dose of Marek's vaccine was similar to that of untreated and non-challenged controls. In summary, the immunomodulators increased hatchability under challenge conditions, while co-administration of the Marek's vaccine enhanced the protective response of the immunomodulators. The results indicate that administration of the immunomodulator or immunomodulator and Marek's vaccine elicits a non-antigen specific immune response.

Example 4. administration of an immunomodulator elicits a non-antigen specific immune response, which is enhanced by additional administration of a biological agent.

A study was conducted to determine the effect of immunomodulators in Marek's disease vaccinated chickens (exposed to e. The study included 9 groups, with different treatments as shown in table 4.

Table 4 study group treatment.

Commercial broiler eggs were incubated for 18 days, checked for viability by light, and then 18-day-old embryonated eggs that received immunomodulators were injected in ovo. The next day, 0.15 mL of each 19-day-old embryonated egg in the challenge group was sprayed to a concentration of 2.63X 10⁵Inoculum of E.coli per ml. Eggs hatch on study day 0, which extends for 45 days.

The percentage of embryonated eggs that hatched from each group was calculated (fig. 24). In hatched chicks, the percent mortality was calculated for the challenged (fig. 26) and non-challenged (fig. 25) groups on day 7 post hatch. The challenged group showed a higher percentage of mortality than the similarly treated non-challenged group (fig. 27). The percentage of surviving chicks of each group was calculated from hatch to day 7 (fig. 28) and day 3 (18 days surviving embryos) to day 7 (fig. 29, n = 400). There was significantly higher survival (> 98.2%, fig. 28) for birds treated with immunomodulators prior to challenge compared to the challenged control group (82.2%, fig. 28). There was significantly higher survival rates at day 7 (95%, fig. 28) for birds treated with immunomodulators at hatch before and after challenge compared to the challenged control group (82.2%, fig. 28). These results were summarized using a set containing 400 eggs per group (fig. 29). There was a higher survival rate for pre-challenge treated embryos at day 7 (> 94.3%, fig. 29) compared to the control group (67.0%, fig. 29). Similarly, there was significantly higher survival rates for birds treated in ovo with immunomodulators on day 14 (97.4%, fig. 30, without second treatment) compared to the challenged control (81.9%, fig. 30). There were significantly higher surviving birds at day 14, 93.9% (fig. 30), without first treatment, in the group treated with immunomodulator at 1 day of age post challenge compared to the control group of 81.9% (fig. 30). There were also significantly higher surviving birds, 95.8% at day 14 in the group treated with the immunomodulator in ovo and subsequently at 1 day of age with the immunomodulator, compared to 81.9% in the control group (fig. 30). Figure 30 shows the weekly mortality of the challenged and non-challenged groups with different treatments.

The data show that there is no reduction in hatchability between the immunomodulator-treated group (treated once or twice) and the untreated group (not challenged with e. Hatchability was higher in the group treated with the immunomodulator before the E.coli challenge. This efficacy is enhanced when the immunomodulator is co-administered with Marek's vaccine. Furthermore, in the non-challenged group, immunomodulatory treatment was associated with decreased mortality. Co-administration of Marek's vaccine and pre-treatment with an immunomodulator prior to administration of the immunomodulator and Marek's vaccine will further reduce mortality. These results are summarized from the attack group. In summary, the immunomodulators increased hatchability under challenge conditions, while co-administration of the Marek's vaccine enhanced the protective response of the immunomodulators. The results indicate that when the administration of the immunomodulator is accompanied by Marek's vaccine, the non-antigen specific immune response elicited by immunomodulator administration is enhanced. Co-administration of immunomodulators with Marek's vaccine further enhances non-antigen specific immune responses.

Example 5 post-infection administration of immunomodulators elicited non-antigen specific immune responses, which were enhanced by additional administration of biological agents.

There was significantly higher survival rates for birds treated with immunomodulators after challenge (95%, fig. 28) compared to the challenged control group (82.2%, fig. 28). These results were summarized using a set containing 400 eggs per group (fig. 29).

The results show that when the administration of immunomodulators is accompanied by Marek's vaccine, the non-antigen specific immune response elicited by the administration of immunomodulators after e. Post-administration of immunomodulators after the Marek's vaccine enhances non-antigen specific immune responses.

Example 6 administration of immunomodulators with bivalent biologies does not inhibit early replication of bivalent biologies

Studies were conducted to determine if immunomodulators would negatively impact early replication of Marek's disease bivalent vaccines. Marek's disease bivalent vaccines include HVT (herpes virus of turkeys) and SB1 (herpes virus of chickens).

Commercial broiler eggs were incubated for 18 days and checked for viability by light. Eggs were divided into 3 groups, a-C20 eggs each. Each egg was inoculated as shown in table 5.

TABLE 5 treatment.

To evaluate the effect of immunomodulators on vaccine virus replication in vivo, virus re-isolation was performed 7 and 14 days after Splenocyte (SPC) and Peripheral Blood Mononuclear Cells (PBMC) placement.

Since MD vaccine virus infection is known to have variation from bird to bird at the level of virus replication, re-isolation was performed using triple pools of chickens (2 chickens per pool, 3 pools per sampling point).

Despite the variation between pools, there was a clear trend in the replication of HVT and SB-1 in the presence of immune modulators. This data was most significant for SB-1 replication in the first week, but the general trends for HVT and SB-1 were similar in both tissues at both time points (day 7 and 14 post-ovalization). The data are summarized in fig. 31 to 38. Figure 31 shows the effect of week 1 immunomodulators on MD vaccine replication of HVT in splenocytes. Figure 32 shows the effect of week 1 immunomodulators on MD vaccine replication of HVT in PBMC. FIG. 33 shows the effect of week 1 immunomodulators on MD vaccine replication of SB-1 in splenocytes. FIG. 34 shows the effect of week 1 immunomodulators on MD vaccine replication of SB-1 in PBMCs. Figure 35 shows the effect of week 2 immunomodulators on MD vaccine replication of HVT in splenocytes. Figure 36 shows the effect of week 2 immunomodulators on MD vaccine replication of HVT in PBMC. FIG. 37 shows the effect of week 2 immunomodulators on MD vaccine replication of SB-1 in splenocytes. FIG. 38 shows the effect of week 2 immunomodulators on MD vaccine replication of SB-1 in PBMCs.

The immunomodulator does not have a negative effect on MD vaccine replication. The immunomodulator appeared to have adjuvant efficacy, increasing replication of HVT and SB-1 in the first two weeks after infection. These data indicate that immunomodulators have a positive effect on MD vaccine efficacy.

Example 7 administration of an immunomodulator with a modified live biological agent does not interfere with the modified live biological agent

Studies were performed to determine the effect of immunomodulators and modified live newcastle disease vaccinated chickens (exposed to newcastle disease). The study included 9 groups, with different treatments as shown in table 6.

Table 6 study group treatment.

Commercial broiler eggs were incubated for 18 days and checked for viability by light. Eggs were divided into 9 groups of 60 eggs each, T1-T9. Group T9 was attacked by the pests the first night after hatch and there were some deaths of unknown cause during the study. The number of birds in each group at the time of picking (culling) is listed in table 7.

Table 7 birds from each group.

Groups T4-T6 were injected with immunomodulators in ovo at 18-day-old embryonated eggs. Groups T1-T3 and T7-T9 injected with saline. On day 0, eggs and chicks hatched in groups T1-T2 were sprayed with saline, groups T3-T6 received Newnotch-C2 (manufactured by Intervet/Schering-Plough Animal Health), and groups T7-T9 were sprayed and inoculated with immunomodulators.

Sera were obtained at 7, 14 and 21 days post-hatch for determination of anti-NDV titers. On day 21, groups of chicks were challenged with a slow-onset newcastle disease virus T2-T9. Chicks were picked on day 26, bled, examined for gross pathology of airsacculitis, and seroanalyzed.

The data show that there were no serologically significant differences between the groups at days 7, 14 and 21. However, on day 26 (5 days post challenge), birds receiving the immunomodulator in ovo had significantly increased anti-NDV titers over all other groups (including vaccine only) (fig. 40).

Air sacculitis was significantly reduced in T2-T5 and these chicks received in ovo modulators (fig. 39). The results indicate that in ovo administration of the immunomodulator does not interfere with NDV vaccination.

Example 8 efficacy of administration of immunomodulators and biologics in toxic challenge

A study was conducted to identify interference of a single dose of immunomodulator on conventional Marek's disease vaccination in 18-day-old embryonated eggs. This study compared the effect of Marek's disease vaccination against toxic challenge.

A total of 160 embryonated eggs were divided into 8 groups. On study day 0, each bird that hatched freshly dehulled was inoculated with Marek's disease virus. The number of hatched uncoating birds was 148 out of 160 (see table 8 below, starting number of eggs/birds vs. actual birds at the end of the study in brackets). Three weeks later, 80 birds, 15 non-vaccinated and untreated birds (contact) and 65 treated birds (inoculation) were added each (see table 8 below, treatment of each group).

TABLE 8 treatment.

On study day 42, surviving unshelled birds were removed and necropsy performed. On study day 63 (day 42 of exposure and inoculation), surviving study birds were removed and necropsy performed.

Fig. 41 shows survival curves of vaccinated, unshelled, non-vaccinated, contact, vaccinated only, and vaccinated/immune-modulator birds. The non-inoculated exposure data are the mean of 8 henhouses, whereas each inoculated group is the mean of 4 henhouses each. It was observed that there was similarity in the slope of the survival curves for vaccinated and vaccinated/immunomodulator birds alone.

FIG. 42 shows the average incidence of Marek's disease in vaccinated, non-vaccinated, contact, vaccinated only, vaccinated/immunomodulator birds. The shelling and exposure data are the mean of 8 henhouses and each inoculum is the mean of 4 henhouses each. It can be observed that the vaccination only group and the vaccination/immunomodulator group had a much lower incidence of Marek's disease. The vaccinated/immunomodulator group had a lower incidence of Marek's disease than the vaccinated group alone.

As can be seen from fig. 41 to 42, the immunomodulator had no damaging effect on Marek's disease vaccine protection. In fact, it elicits an increase in vaccine efficacy.

Claims (6)

1. Use of an immunomodulator composition in the preparation of a medicament for increasing the incidence of escherichia coli by administering an effective amount of 0.1 to 10 micrograms of said immunomodulator composition in ovo prior to incubation (Escherichia coli) Use of a medicament for challenging embryonated egg hatchability, wherein the immunomodulator composition comprises

a. A cationic liposome delivery vehicle; and

b. a nucleic acid molecule, wherein the nucleic acid molecule is an isolated nucleic acid vector of bacterial origin, or a fragment thereof, that does not contain a gene insert.

2. The use of claim 1, wherein the liposome delivery vehicle comprises a lipid selected from the group consisting of multilamellar vesicle lipids and extruded lipids.

3. The use of claim 1 or 2, wherein the liposome delivery vehicle comprises a pair of lipids selected from DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol.

4. The use of claim 1 or 2, wherein the immunomodulator composition further comprises a biological agent, and wherein the biological agent is a vaccine for protection against a virus or disease selected from the group consisting of: marek's Disease Virus (MDV), Newcastle Disease Virus (NDV), Chicken Anemia Virus (CAV), Infectious Bursal Disease Virus (IBDV), Infectious Bronchitis Virus (IBV), turkey Herpes Virus (HVT), infectious laryngotracheitis virus (ILTV), Avian Encephalomyelitis Virus (AEV), fowlpox virus (FPV), fowl cholera, Avian Influenza Virus (AIV), reovirus, Avian Leukosis Virus (ALV), reticuloendotheliosis virus (REV), avian adenovirus, Hemorrhagic Enteritis Virus (HEV), and combinations thereof.

5. The use of claim 1 or 2, wherein the cationic liposome delivery vehicle is a combination of DOTIM and cholesterol.

6. The use of claim 1 or 2, wherein the administration is prior to challenge.

7The use of claim 1 or 2, wherein the immunomodulator composition is administered to an embryonated chicken egg on day 18 of incubation.

8The use of claim 1, wherein the effective amount is 0.1 to 5 micrograms/egg.

9The use of claim 8, wherein the effective amount is 1.0 micrograms/egg.