Title:  Adjuvanted vaccine which is substantially free of non-host albumin

United States Patent:  6,682,746

Issued:  January 27, 2004

Inventors:  Hennessy; Kristina J. (Bayer Corporation, 100 Bayer Rd., Pittsburgh, PA 15205-9741); Brown; Karen K. (Bayer Corporation, 100 Bayer Rd., Pittsburgh, PA 15205-9741); Lane; Jennifer K. (Bayer Corporation, 100 Bayer Rd., Pittsburgh, PA 15205-9741); Trump; Sandra L. (Bayer Corporation, 100 Bayer Rd., Pittsburgh, PA 15205-9741)

Appl. No.:  099182

Filed:  March 14, 2002

Abstract

Disclosed herein is a serum-based adjuvanted vaccine which is substantially free of non-host albumin and the use thereof in reducing or preventing post-vaccination systemic reactions.

SUMMARY OF THE INVENTION

In accordance with the foregoing, the present invention encompasses a serum-based vaccine comprising an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin. The term "serum-based" is used herein to denote that the vaccines of the invention or their precursors employ serum including non-host serum. Typically, the serum is employed in growth media to enhance growth of organisms that are employed in the preparation of the vaccine. By the term "precursor of the vaccine" is meant vaccine components, particularly antigen, proteins other than antigen, whole organisms and harvest material. By the term "immunogenically effective amount" is meant that the antigen contains a protective component in a concentration that is sufficient to protect animals from a target disease when an adjuvanted vaccine containing the antigen is administered to animals. By the term "antigen" is meant a biological material (natural, recombinant or synthetic) that stimulates a protective immune response in animals. By the term "adjuvanted vaccine" is meant a vaccine containing an adjuvant, or a plurality of vaccines administered as a part of a vaccine regimen wherein at least one of the vaccines contains an adjuvant. By the term non-host albumin is meant albumin from the serum of an animal species other than the animal species being vaccinated. Albumin is a simple protein found in serum and has a molecular weight of about 66,000 daltons. A vaccine which is substantially free of non-host albumin contains less than 1.0 mg/mL of non-host albumin.

Also encompassed by the invention is a method of preparing the serum-based vaccine that is substantially free of non-host albumin comprising removing non-host albumin from the vaccine or a precursor thereof. An alternate method of preparing the serum-based vaccine that is substantially free of non-host albumin comprises providing a host serum containing host albumin in the preparation of the vaccine.

Further encompassed by the invention is a vaccine which is prepared by adding host serum or albumin to the vaccine antigen after harvesting or purifying the antigen from a culture of an organism from which the antigen is derived, but prior to adjuvanting the antigen. Additionally, the host serum or albumin can be added to the antigen after harvesting but prior to lyophilizing the antigen if the antigen is a modified live organism. When host serum or host albumin is used in this manner, it acts as a stabilizer. The term "stabilizer" means any additive that is added to a vaccine to prevent degradation of the antigen and the consequential loss of immunogenicity of the vaccine.

In a presently preferred embodiment of the invention, the method of preparing a serum-based vaccine containing an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin comprises:

(a) growing an organism which produces the antigen in a culture containing non-host albumin;

(b) harvesting the culture;

(c) clarifying the harvest;

(d) separating the antigen and non-host albumin from the clarified harvest;

(e) separating the non-host albumin from the antigen;

(f) collecting the antigen; and

(g) formulating the antigen with an adjuvant.

In an additional preferred embodiment of the invention, the method of preparing a serum-based vaccine containing an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin comprises:

(a) growing an organism which produces the antigen in a culture containing non-host albumin;

(b) harvesting the culture;

(c) clarifying the harvest;

(d) separating the antigen from the non-host albumin by passing the clarified harvest through a column with a matrix which selectively binds the antigen;

(e) washing the column matrix to remove excess non-host albumin;

(f) discarding the wash solution;

(g) washing the column matrix with a solution which elutes the antigen from the column matrix;

(h) collecting the antigen; and

(i) formulating the antigen with an adjuvant.

In another preferred embodiment of the invention, the method of preparing a serum-based vaccine containing an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin comprises:

(a) growing an organism which produces the antigen in a culture containing non-host albumin;

(b) harvesting the culture;

(c) clarifying the harvest;

(d) separating the antigen from the non-host albumin by passing the clarified harvest through a column with a matrix which selectively binds the non-host albumin;

(e) collecting the antigen; and

(f) formulating the antigen with an adjuvant.

In still another preferred embodiment of the invention, the method of preparation of a serum-based vaccine containing an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin comprises:

(a) growing an organism which produces the antigen in a culture containing host albumin;

(b) harvesting the culture;

(c) clarifying the harvest, if necessary; and

(d) formulating the harvest with an adjuvant.

Further encompassed by the invention is a method of eliminating adverse vaccine reactions in animals comprising administering to said animals a vaccine regimen which is substantially free of non-host albumin.

The method for eliminating adverse reactions in animals comprises administering to said animals an adjuvanted vaccine or an adjuvanted vaccine regimen which is substantially free of non-host albumin.

Also encompassed by the invention is a process for stabilizing an antigen comprising adding host serum or host albumin to said antigen prior to adjuvanting the antigen. Such a process for stabilizing an antigen can also comprise adding host serum or host albumin to said antigen prior to lyophilizing the antigen.

The vaccines of the invention are applicable for use in preventing or treating diseases of all species of animals. They are particularly suitable for use in preventing or treating diseases of companion animals such as cats, dogs and horses which are particularly sensitive to adjuvanted vaccine regimens comprising non-host albumin. In particular, the vaccines of the invention are suitable for use in preventing feline leukemia (FeLV) and rabies because they are free of problems that typically attend such vaccines FeLV vaccines are notorious for causing adverse reactions such as hypersalivation, vomiting, diarrhea and sometimes death. Often, these reactions occur within minutes of administration of the vaccine.

Surprisingly, it has been found that animals to which the vaccines of the invention have been administered have virtually no adverse systemic reactions. The discovery that non-host albumin in a vaccine containing an adjuvant or administered in a vaccine regimen with a vaccine containing an adjuvant can cause systemic reactions is thus a part of the invention. This and other aspects of the invention are described more fully hereunder.

DETAILED DESCRIPTION OF THE INVENTION

As set forth above, the present invention encompasses a serum-based vaccine comprising an immunogenically effective amount of an antigen and an adjuvant wherein the vaccine is substantially free of non-host albumin and methods of making and using the same. It also encompasses a vaccine regimen wherein at least one vaccine in the regimen contains an adjuvant and at least one vaccine in the regimen contains non-host albumin. In addition, it encompasses a process for stabilizing an antigen comprising adding host serum or host albumin to said antigen prior to adjuvanting the antigen. Such a process for stabilizing an antigen can also comprise adding host serum or host albumin to said antigen prior to lyophilizing the antigen.

Non-host albumin is derived from non-host serum that is typically used in growing organisms from which the antigens are derived. Typical examples of non-host serum (containing non-host albumin) can be selected from the group consisting of bovine serum, fetal bovine serum, equine serum, fetal equine serum, sheep serum and goat serum. On the other hand, if equine albumin is present in an equine vaccine, the vaccine is considered to contain host albumin.

The antigen is obtained from an organism selected from the group consisting of bacteria, virus, parasite, rickettsia and protozoa. Examples of the bacteria can be selected from the group consisting of Bordetella spp., Streptococcus spp., Staphylococcus spp., Clostridium spp., Leptospira spp., Escherichia spp., Salmonella spp., Pasteurella spp., Mycobacteria spp., Mycoplasma spp., Moraxella spp., Haemophilus spp., Borrelia spp., Fusobacteria spp., Bacteriodes spp. and Rhodococcus spp. Examples of the viruses can be selected from the group consisting of herpes viruses, parainfluenza viruses, reoviruses, rotaviruses, morbilliviruses, retroviruses, coronaviruses, adenoviruses, togaviruses, parvoviruses, parapox viruses, paramyxoviruses, cytomegaloviruses, arboviruses and hantaviruses. More specifically, such viruses would include but not be limited to feline leukemia virus, feline rhinotracheitis, feline calicivirus, feline panleukopenia virus, feline immunodeficiency virus, feline infectious peritonitis virus, canine hepatitis, canine adenovirus type 2, canine parvovirus, rabies virus, canine parainfluenza virus, canine coronavirus, equine herpes viruses, equine influenza viruses and equine encephalomyelitis viruses. Examples of parasites and protozoa can be selected from the group consisting of Neospora spp., Toxoplasma spp., Dirofilaria spp., Cryptosporidium spp., Giardia spp., Babesia spp, and Coccidia spp. An example of rickettsia can be selected from the group consisting of Chlamydia spp., Potomac Horse Fever, Ehrlichia canis, and other Ehrlichia spp.

The antigens can be obtained from a member selected from the group consisting of: a whole culture of an organism such as a whole culture harvest, a partially purified whole culture harvest, a purified subunit extracted from harvest, a subunit obtained via recombinant technology and expressed in the homologous or a heterologous organism, a deletion mutant of the whole organism (conventional or rDNA gene-deleted mutants), peptides, naked DNA, chemically synthesized antigens, reverse transcribed naked cDNA or combinations thereof.

Generally, the antigen can be produced by art-known techniques of culturing and harvesting organisms, concentrating and/or conventionally purifying antigens of such organisms. For example, the antigen can be produced by: growing the selected organism in a culture having growth medium containing a non-host serum (serum-based culture). More specifically, the organism can be grown in a tissue culture prepared from mammalian or plant cells wherein non-host serum is added to the medium to enhance the growth of the organism. The organism can also be grown in fermentation media wherein the organism grows without tissue culture but has added thereto a growth medium containing a non-host serum. Typically, the non-host serum can be selected from the group consisting of fetal bovine serum, bovine serum, calf serum, fetal equine serum, horse serum, goat serum, lamb serum and sheep serum. At the completion of growth, the culture is harvested and, if necessary, conventionally purified by, say, filtration and/or ultrafiltration to remove cells, cellular debris and extraneous contaminants. However, these techniques do not remove the non-host albumin. At this point, the culture harvest still contains non-host albumin and would not be acceptable if combined with adjuvant and/or administered in a regimen with an adjuvanted vaccine. Therefore, the resulting culture harvest is further purified in accordance with this invention to remove the non-host albumin prior to its formulation into an adjuvanted vaccine.

In accordance With the invention, the non-host albumin can be removed by a process of purifying the vaccine or a precursor of the vaccine in such a manner as would remove the non-host albumin. The process of purifying the precursor of the vaccine can be done by a chromatography technique selected from the group consisting of PERFUSION CHROMATOGRAPHY.RTM. (PerSeptive Biosystems), ion exchange chromatography, molecular sieve chromatography, hydrophobic interaction chromatography, affinity chromatography and combinations thereof. Preferably, the process of purification is by PERFUSION CHROMATOGRAPHY.RTM. using hydrophobic interaction chromatography matrices or a combination of hydrophobic interaction chromatography and ion exchange chromatography. The following is an illustrative but non-limiting description of the hydrphobic interaction chromatography with a PERFUSION CHROMATOGRAPHY.RTM. matrix utilizing POROS media (PerSeptive Biosystems).

PERFUSION CHROMATOGRAPHY.RTM. is carried out using a matrix (POROS media) having large channeled pores which carry molecules swiftly into the interior of each bead by convective flow as well as diffusive pores that branch off the channeled pores providing a large internal surface area for binding. This pore combination provides high capacity, high resolution and high-speed purification. Hydrophobic interaction chromatography involves the use of polar groups on an uncharged matrix to interact with polar residues (e.g. phenlyalanine) on proteins, causing retardation and separation of proteins based on their relative hydrophobicites. The use of the POROS media matrix allows much greater flow rates at higher pressures so that the purification time is reduced, thus reducing the cost and allowing chromotography to be cost effective for veterinary products.

Hydrophobic interaction chromatography is performed by adding a high ionic strength buffer to fluids of the culture harvest containing the non-host albumin before adding such fluids to the hydrophobic column. The column is washed several times with a high ionic strength buffer such as 20 millimolar (Mm) sodium phosphate/650 Mm sodium sulfate before addition of the high ionic strength buffered fluids of the culture harvest containing the non-host albumin (column feed material). Multiple column volumes of column feed material are run through the column. The column matrix binds both the non-host albumin and the antigen (contained within the buffered fluids of the culture harvest). To elute non-host albumin from the column, the column is washed multiple times with a high ionic strength buffer such as 20 Mm sodium phosphate/650 Mm sodium sulfate or until the optical density reading at a wavelength of 280 nanometers (nm) of the eluate is less than 0.03. The antigen (purified) is eluted from the column by washing the column matrix with multiple volumes of a low ionic strength solution which can be sterile water. The purified antigen is collected in a separate collecting vessel when the optical density of the eluate increases above 0.15. Collection of the eluate ceases when the optical density of the eluate drops below 0.10.

Another method for removal of non-host albumin according to this invention encompasses use of affinity chromatography for binding of either the antigen or the non-host albumin. For instance, the antigen can be produced by art-known techniques of culturing and harvesting organisms and clarigyin, concentrating and/or conventionally purifying antigens of such organisms as described previously. For removal of the non-host albumin the clarified harvest can be added to a columns containing a matrix which binds either the antigen or which binds the non-host albumin. Such a matrix could be a lectin such as CIBACHRON.TM. Blue (Pharmacia) or Mimetic Blue (Affinity Chromatography Ltd.), both of which bind non-host albumin, or a matrix which contains a polyclonal or monoclonal antibody specific for the antigen or non-host albumin, whichever is to be bound to the matrix. The clarified harvest becomes the column feed material and is added to the column. If the column contain a matrix such as a lectin, a polyclonal antibody or a monoclonal antibody specific for non-host albumin, the non-host albumin is bound to the column and the antigen passes through the column and is collected. The collected antigen is then formulated with adjuvant to prepare a vaccine. If the column contains a matrix such as a plyclonal antibody or monoclonal antibody specific for antigen, the clarified harvest material is added to the column and the antigen is bound to the matrix. The non-host albumin passes through the column and is discarded. Excess non-host albumin is removed from the column matrix by washing with a buffer which does not remove the antigen. Then the matrix is washed with a solution which elutes the antigen from the column matrix. Such washing and elution buffers can be based on pH, ionic strength or polarity differences of the antigen to be eluted. The antigen is then collected and formulated with an adjuvant to produce the vaccine. If a lectin is used to bind non-host albumin, the antigen which is collected will have to be further purified through a second lectin column or by using another type of chromatography to remove all of the non-host albumin.

If one has a whole organism such as a virus or bacteria or a very large antigen, for instance, one with a molecular weight greater than 100,000 daltons, molecular sieve chromatography can be used to separate the antigen from the non-host albumin which has a molecular weight of only about 66,000 daltons. Molecular sieve chromatography separates molecules on the basis of molecular weight. The matrix is selected so that low molecular weight molecules such as non-host albumin pass through the column at a faster rate than large molecular weight molecules such as large antigens. Using this technique, the organism is grown in a culture containing non-host albumin and harvested, clarified and/or concentrated and purified by conventional techniques as described previously. In order to separate the non-host albumin from, for instance, a whole virus, the virus is grown in tissue culture, harvested by collecting the fluids from the tissue culture and clarified to remove the cellular debris. This clarified harvest is the column feed material and is added to the column. The first fluid to pass through the column is collected and discarded since it contains the non-host albumin. The virus passes through the column slower and can be washed into a collection vessel using buffers which do not harm the virus. Virus which has been collected in this manner can be formulated with an adjuvant to prepare a vaccine.

An alternate method of preparing the serum-based vaccine containing an immunogenically effective amount of an antigen and an adjuvant wherein said vaccine is substantially free of non-host albumin comprises culturing the organism in host serum wherein there is no non-host albumin. By this method, one grows the organism in tissue culture or fermentation media containing host serum instead of non-host serum. Conventional harvesting, concentration and purification can be used if a pure product is desired. No further purification to remove non-host albumin is required because the preparation does not contain non-host albumin. By this method, the crude harvest material can also be used to formulate the vaccine. Using this method the harvest material can simply be combined with adjuvant to formulate the vaccine.

Following the purification and/or removal of the non-host albumin or growth of the organism in host serum, the antigen is inactivated and adjuvanted by conventional techniques. Generally stated, the antigen can be inactivated by treating it with an inactivating agent which does not denature the protective component of the antigen. Specifically, the antigen can be inactivated by treating it chemically, by irradiation, by heating or by freeze-thaw. Illustratively, one can employ chemical inactivating agents selected from the group consisting of formalin, beta-propiolactone, detergents and binary ethyleneimine. Different ones of these chemical inactivating agents are preferred for different organisms.

The inactivated antigen can also be concentrated or pooled with other harvested antigen prior to adjuvanting. The amount of concentration would be such that the average amount of antigen or Relative Potency (RP) value meets or exceeds the minimum acceptable value for a vaccine. The inactivated antigen may be concentrated up to 100 fold, if necessary, by ultrafiltration with a molecular weight cut-off which will suitably maintain the antigen and allow contaminants to pass through and be discarded or by differential centrifugation. After inactivation, the antigen value must be above the acceptable minimum level or RP. Then it is stored at temperatures from -70^oC. to +10^oC. until it is mixed or microfluidized with an adjuvant.

The inactivated antigen is formulated or combined with an adjuvant. Adjuvants are chemicals or bacterial or virus-derived components added to vaccines to enhance the production of an immune response by the animal receiving the vaccine. Adjuvants fall into the general categories of polymers, block co-polymers, oils, oil-in-water, aluminum salts, and bacterial and viral extracts. Most adjuvants function by producing an irritation at the site of injection causing leukocytes (immune cells) to infiltrate the area and/or by producing a depot effect (holding the antigens at the injection site for as long as possible). Some of the newer adjuvants act as slow-release mechanisms, releasing antigens encapsulated by them at a relatively slow rate. Even newer adjuvants directly affect the B-cells or T-cells of the immune system and are called immune stimulators, immune regulators, immune modulators or immune enhancers. If an adjuvant causes extensive infiltration of leukocytes to the injection site, swelling and injection-site reactions will occur. The immune response to adjuvants may also enhance the reactivity to contaminants such as endotoxins, thereby increasing the probability of systemic reactions such as anaphylaxis. Therefore, although adjuvants are necessary for stimulation of the immune response by inactivated vaccines, they can produce detrimental side effects. The adjuvant is selected from the group consisting of polymers, block co-polymers, oils, oil-in-water, water-in-oil, aluminum salts, immuno-modulators and combinations thereof. Preferably, the adjuvant is a polymer or block co-polymer. The adjuvant can be employed in an amount of from 0.01% to 50%. The amount of adjuvant is strictly correlated to the type of adjuvant used. However, it is important that the adjuvant be employed in an effective amount to immunogenically stimulate the inactivated antigens. When used in such an amount, adjuvants can stimulate adverse reactions to non-host albumin.

After inactivating and adjuvanting the antigen, the potency or Relative Potency (RP) of the antigen can be adjusted to an appropriate level which meets or exceeds the minimum acceptable amount of antigen to produce an immunogenically effective vaccine. The tests used for such potency or relative potency testing are described hereunder. The antigen(s) can be formulated with other antigens. For example, inactivated and adjuvanted feline leukemia virus prepared in accordance with the invention can be formulated with feline calicivirus, feline panleukopenia virus, feline rhinotracheitis virus and feline chlamydia. Additionally, inactivated and adjuvanted rabies virus prepared in accordance with the invention can be formulated with canine parvovirus, canine distemper virus, canine parainfluenza virus, canine adenovirus type 2 and various Leptospira spp. Also, inactivated and adjuvanted equine viruses and bacterial antigens can be prepared in accordance with the invention. Some or all of these additional antigens may be prepared according to the present invention. Some of the additional antigens may be modified live. However, the final combination vaccines will be substantially free of non-host albumin if the combination vaccine or vaccine regimen wherein the combination vaccine is administered contains an adjuvant. The resulting adjuvanted vaccine that is substantially free of non-host albumin is safe and effective and can be administered to animals with essentially no post-vaccination, adverse systemic reactions.

As a measure of vaccine potency that equates to vaccine protection in the host animal, each individual lot of antigen (crude or purified) and serial of vaccine undergoes testing. The measurement may involve vaccination of laboratory animals or host animals followed by a challenge of the animals, vaccination of laboratory animals or host animals followed by evaluation of a serological response or the performance of an Enzyme Linked Immunosorbant Assay (ELISA) to measure the amount of antigens in the vaccine. An Enzyme Linked Immunosorbant Assay (ELISA) is preferable as it eliminates animal testing. In the latter method, the antigen concentration in the test vaccine is measured against the antigen content in a Reference Vaccine which has been proven to be protective in the host animal. A test vaccine which measures 1.0 as compared with the Reference Vaccine is considered to be potent and is said to have a relative potency (RP) of 1.0. The RP can be measured before or after the antigen has been harvested, purified, inactivated or adjuvanted. Before inactivation and adjuvanting, the RP must be above 1.0 so that after inactivation and adjuvanting it does not fall below 1.0.

The purified antigen in accordance with the invention, may be concentrated or pooled with other purified harvested antigen such that the average amount of antigen meets or exceeds the minimum acceptable value for a harvest. The purified antigen may be concentrated up to 100 fold, if necessary, by ultrafiltration with a molecular weight cut-off which will suitably maintain the antigen and allow contaminants to pass through and be discarded or by differential centrifugation. It is important to note that even if very low levels of serum are used for growth enhancement, it is virtually impossible to remove its albumin content from cultures of the organism or vaccines by conventional purification processes, especially if concentration is used. For instance, if antigen is concentrated 100 fold, a non-host albumin level of 0.1% (1 mg/mL) in the antigen prior to concentration would be concentrated to 10% or 100 mg/mL after concentration. Such a level would be totally unacceptable in the final vaccine.

As would be realized from the foregoing, a distinct feature of the invention is the discovery of the source of the problem of post-vaccination adverse systemic reactions and the solutions for the problem. Without being bound to any particular theory, it is believed that the adverse post-vaccination systemic reactions result from the presence of adjuvants and non-host albumin in vaccines or vaccine regimens. There is hereby discovered and disclosed a solution which includes removing non-host albumin from vaccines which contain an adjuvant or which are administered in vaccine regimens which contain adjuvanted vaccines or using host serum for antigen preparation in place of non-host serum and administering vaccines and vaccine regimens which are substantially free of the non-host albumin.

Claim 1 of 11 Claims

What is claimed is:

1. A method for avoiding adverse systemic reactions in an animal immunized with a serum-based vaccine comprising:

(a) culturing an antigen in a non-host serum culture medium,

(b) removing substantially all of the non-host albumin from said antigen before formulating the vaccine,

(c) formulating the vaccine which comprises an immunogenically effective amount of a serum-grown antigen from step (b) and an adjuvant and

(d) administering an immunogenically effective amount of the vaccine to the animal.

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