The present invention relates generally to immunogenic agents and to agents which enhance the immune response to a selected antigen. In particular, the invention pertains to the use of protein particles as antigens to elicit cellular immune responses.
Numerous vaccine formulations which include attenuated pathogens or subunit protein antigens, have been developed. Conventional vaccine compositions often include immunological adjuvants to enhance cell-mediated and humoral immune responses. For example, depot adjuvants are frequently used which adsorb and/or precipitate administered antigens and which can retain the antigen at the injection site. Typical depot adjuvants include aluminum compounds and water-in-oil emulsions. However, depot adjuvants, although increasing antigenicity, often provoke severe persistent local reactions, such as granulomas, abscesses and scarring, when injected subcutaneously or intramuscularly. Other adjuvants, such as lipopolysacharrides, can elicit pyrogenic responses upon injection and/or Reiter""s symptoms (influenza-like symptoms, generalized joint discomfort and sometimes anterior uveitis, arthritis and urethritis). Saponins, such as Quillaja saponaria, have also been used as immunological adjuvants in vaccine compositions against a variety of diseases.
More particularly, Complete Freund""s adjuvant (CFA) is a powerful immunostimulatory agent that has been successfully used with many antigens on an experimental basis. CFA includes three components: a mineral oil, an emulsifying agent, and killed mycobacteria, such as Mycobacterium tuberculosis. Although effective as an adjuvant, CFA causes severe side effects primarily due to the presence of the mycobacterial component, including pain, abscess formation and fever. CFA, therefore, is not used in human and veterinary vaccines.
Incomplete Freund""s adjuvant (IFA) is similar to CFA but does not include the bacterial component. IFA, while not approved for use in the United States, has been used elsewhere in human vaccines for influenza and polio and in veterinary vaccines for rabies, canine distemper and foot-and-mouth disease. However, evidence indicates that both the oil and emulsifier used in IFA can cause tumors in mice.
Despite the presence of such adjuvants, conventional vaccines often fail to provide adequate protection against the targeted pathogen. In this regard, there is growing evidence that vaccination against intracellular pathogens, such as a number of viruses, should target both the cellular and humoral arms of the immune system. More particularly, cytotoxic T-lymphocytes (CTLs) play an important role in cell-mediated immune defense against intracellular pathogens such as viruses and tumor-specific antigens produced by malignant cells. CTLs mediate cytotoxicity of virally infected cells by recognizing viral determinants in conjunction with class I MHC molecules displayed by the infected cells. Cytoplasmic expression of proteins is a prerequisite for class I MHC processing and presentation of antigenic peptides to CTLs. However, immunization with killed or attenuated viruses often fails to produce the CTLs necessary to curb intracellular infection. Furthermore, conventional vaccination techniques against viruses displaying marked genetic heterogeneity and/or rapid mutation rates that facilitate selection of immune escape variants, such as HIV or influenza, are problematic. Accordingly, alternative techniques for vaccination have been developed.
Particulate carriers with adsorbed or entrapped antigens have been used in an attempt to circumvent these problems and in attempts to elicit adequate immune responses. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes. The particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as polymer particles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. While offering significant advantages over other more toxic systems, antigen-containing PLG particles suffer from some drawbacks. For example, large scale production and manufacturing of particulate carriers may be problematic due to the high cost of the polymers used in the manufacture the particulate carriers.
Liposomes have also been employed in an effort to overcome these problems. Liposomes are microscopic vesicles formed from lipid constituents such as phospholipids which are used to entrap pharmaceutical agents. Although the use of liposomes as a drug delivery system alleviates some of the problems described above, liposomes exhibit poor stability during storage and use, and large scale production and manufacturing of liposomes is problematic.
International Publication No. WO 98/50071 describes the use of viral-like particles (VLPs) as adjuvants to enhance immune responses of antigens administered with the VLPs. St. Clair et al. describe the use of protein crystals to enhance humoral and cellular responses. (St. Clair, N. et al, Applied Biol. Sci., 96:9469-9474, 1999).
Despite the above described adjuvant and antigen-presentation systems, there is a continued need for effective, safe and cost-efficient vaccines with improved purity, stability and immunogenicity.
The inventors herein have found, surprisingly, that protein particles are self-sustaining immunogenic agents which produce cellular immune responses. In particular, the active ingredient is also the delivery system, i.e., the protein particles serve as the antigen and the delivery system. Additionally, the inventors have discovered that the protein particles have several advantages (i) the ease of manufacture, (ii) they are more cost-effective to manufacture than existing agents, (iii) they provide for superior immune responses, and (iv) they have reduced toxicity and eliminate the undesirable side-effects observed with other vaccine formulations. Accordingly, then, the invention is primarily directed to the use of such protein particles as antigens.
In one embodiment, the invention is directed to an immunogenic composition comprising selected first antigen and a pharmaceutically acceptable excipient, wherein the selected first antigen is a protein particle, and further wherein the protein particle antigen is capable of producing a cellular immune response. In preferred embodiments, the protein particle is formed from a protein selected from the group consisting of a viral, a fungal, a bacteria, an avian or a mammalian protein. In more preferred embodiments, the protein is herpes simplex virus type 2 glycoprotein B (HSV gB2), hepatitis C virus (HCV) or a human immunodeficiency virus (HIV) protein.
In another embodiment, the immunogenic composition further comprising an adjuvant, wherein the adjuvant is encapsulated within, adsorbed or conjugated on to, or mixed with the protein particle.
In an additional embodiment, the immunogenic composition further comprises a second antigen, wherein the second antigen is distinct from the first antigen, i.e. the protein particle. The second antigen may be a soluble or neutralizing antigen, it may be conjugated on to the protein particle, or it may be associated with a carrier (for example, the second antigen may be encapsulated within, adsorbed or conjugated on to, or mixed with the carrier). In certain preferred embodiments, the carriers include, but are not limited to proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), polymeric particulate carriers, and inactive virus particles. In more preferred embodiments, the carriers comprise a poylmeric particle, wherein the polymeric particle comprises a polymer selected from the group consisting of a poly(xcex1-hydroxy acid), a polyhydroxy butyric acid, a polycaprolactone, a polyorthoester, and a polyanhydride.
In an alternative embodiment, the invention is directed to an immunogenic composition comprising a selected first antigen and a pharmaceutically acceptable excipient, wherein the selected first antigen is a protein particle, and further wherein the protein particle is produced by a process comprising the steps of:
(a) providing an aqueous solution of a protein;
(b) adding a precipitation agent to the aqueous solution of the protein and stirring the resulting mixture to form the protein particle;
(c) stabilizing said protein particle by a stabilizing treatment; and
(d) recovering the protein particles from the aqueous solution.
In an alternative embodiment, the aqueous solution is step (a) further comprises an acid, wherein the acid is acetic acid, glycolic acid, hydroxybutyric acid, hydrochloric acid or lactic acid. In preferred embodiments, the precipitation agent comprises oils, hydrocarbons or coacervation agents. In additional preferred embodiments, the stabilizing treatment comprises heat treatment or by treatment with a chemical cross-linking agent.
In preferred embodiments, the protein particle is capable of producing a cellular immune response; and is formed from a protein selected from the group consisting of a viral, a fungal, a bacterial, an avian or a mammalian protein. In more preferred embodiments, the protein is herpes simplex virus type 2 glycoprotein B (HSV gB2), hepatitis C virus (HCV) or a human immunodeficiency virus (HIV) protein. In certain preferred embodiments, the cellular immune response can be a cytotoxic-T lymphocyte (CTL) response. In another embodiment, the immunogenic or vaccine composition further comprises an adjuvant and/or a second antigen as described above, wherein the protein particle is capable of functioning as an antigen and/or an adjuvant.
In another embodiment, the subject invention is directed to a method for producing a cytotoxic-T lymphocyte (CTL) response in a vertebrate subject comprising administering to the vertebrate subject an immunogenic or vaccine composition comprising the protein particle as described above. The protein particle is administered in an amount effective for eliciting a cytotoxic-T lymphocyte (CTL) response in the vertebrate subject. The protein particles can be co-administered to the subject prior or subsequent to, or concurrent with, an adjuvant and/or a second antigen.
In another embodiment, the invention is directed to a method of immunization which comprises administering to a vertebrate subject a therapeutically effective amount of the immunogenic or vaccine composition comprising the protein particle as discussed above.
These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.