Vaccination is a highly effective medical intervention aimed at reducing the morbidity and mortality caused by infectious diseases in animals, including humans. Vaccination induces an immune response against antigens present in the vaccine that protects against subsequent exposure to infectious agent(s). It has been reported that adsorption of an antigen to aluminum-containing adjuvants may in some cases enhance the immunogenicity of the antigen. It has also been reported that complexation of antigen with adjuvant may in other cases facilitate uptake of antigen in the antigen-presenting dendritic cells (Hem and HogenEsch, Expert Review of Vaccines 2007, 6, 685-698; the preceding publication, and all other publications cited herein, are incorporated herein by reference in their entirety). Thus, adjuvants are substances often added to vaccines to achieve a more effective immune response (Rev. Inf. Dis. 1980, 2, 370; Nat. Rev. Microbiol. 2007, 5, 505).
In particular, aluminum adjuvants are often added. It is appreciated that currently, only aluminum adjuvants have been approved for use in human vaccines by the Food and Drug Administration. Aluminum adjuvants have a long (>60 years) record of safety. The adjuvant aluminum hydroxide (AH) consists of small primary particles less than 50 nm in size that form loose aggregates about 1-20 μm in diameter (Expert Rev. Vaccines 2007, 5, 685). The particulate nature gives the aluminum adjuvant a very large adsorptive capacity. The two main mechanisms of adsorption are electrostatic adsorption and ligand exchange adsorption. The surface of AH contains only hydroxyl groups that give it a positive surface charge at neutral pH, and a high capacity for ligand exchange. Ligand exchange involves the substitution of —OH groups at the surface by phosphate groups present on the antigen, resulting in strong binding of the molecule to which the phosphate group is attached.
For protein antigens that naturally contain phosphorylated amino acids (typically phosphoserine or phosphothreonine) such complexation may be straightforward. In cases where the proteins do not contain phosphate groups, or sufficient phosphate groups, such groups must be introduced in a separate step prior to combining the protein and aluminum hydroxide adjuvant. It has been reported that phosphate groups may be introduced by combining the protein, phosphoserine, and a peptide coupling reagent, such as a carbodiimide, to form a covalent amide bond between the added phosphoserine carboxyl group and amine groups (typically lysine) on the protein. However, it is observed that such a process will also form amide bonds between amino and carboxyl groups on the protein molecules themselves, thus forming protein oligomers, such as dimers, trimers, etc., forming crosslinks within the protein, and generally leading to a complicated mixture of components in the prepared formulation. New reagents and processes are needed for preparing such protein formulations.
In one illustrative embodiment of the invention herein, reagents for preparing immunogenic compositions are described herein. In another embodiment, reagents for attaching one or more phosphates or phosphate mimetic groups to a protein or peptide are described.
In another embodiment, described herein are methods for attaching a protein or peptide to an adjuvant. In another embodiment, described herein are methods for preparing a peptide or protein immunogenic compound or vaccine, where the peptide or protein immunogenic compound or vaccine comprises a complex of the protein or peptide with an adjuvant. In another illustrative embodiment, the adjuvant is an aluminum hydroxide.
In another embodiment, described herein are conjugate molecules composed of three moieties: A phosphate or phosphomimetic group at one end, a spacer or linker, and an active (N-hydroxysuccinimide) ester at the other end. In one illustrative aspect, the active ester is capable of coupling with reactive groups on the protein or peptide, such as on the surface of a protein or peptide (illustratively amino groups, such as but not limited to those on lysine residues in the protein or peptide, and/or hydroxyl or thiol groups, such as but not limited to those on serine, threonine, and cysteine residues in the protein or peptide). Successful coupling of these molecules with a protein or peptide provides one or more phosphate or phosphomimetic groups on the surface of the protein or peptide. Without being bound by theory, it is believed that the “phosphorylation” of the protein or peptide increases the affinity for adjuvants, such as aluminum hydroxide adjuvants, and enhances the utility of the protein or peptide as an immunogenic compound or vaccine. It has been reported that existing strategies to introduce phosphate groups into proteins lead to undesired modifications of the protein, as described herein. The molecules of the invention described herein introduce phosphate or phosphomimetic groups without undesired modification of the protein or peptide.
In another embodiment, conjugates are described herein for increasing the binding of antigens to adjuvants.
In another embodiment, conjugates are described herein for increase the immunogenicity of antigens.
In another embodiment, conjugates and processes for preparing conjugates are described herein for modifying the affinity of antigens for adjuvants. In one aspect, the affinity may be tuned between low, moderate, and high affinity as needed for various configurations of the conjugates, immunogenic compositions, and/or vaccines described herein. Without being bound by theory, it is believed herein that the degree of conjugation or loading is related to the affinity for adjuvants, where a higher affinity is observed when there is higher conjugation loading.
In another illustrative embodiment, described herein is a compound of the formula
wherein
P is a phosphorus containing group;
X is a leaving group;
Q is polyvalent linker;
m is an integer in the range from 1 to about 3; and
n is an integer in the range from 1 to about 20.
In another illustrative embodiment of the compounds described herein, Q is polyvalent heteroalkylene. In another illustrative embodiment, Q is alkyleneamino(alkyl)2, where each alkyl is independently selected. In another illustrative embodiment, m is 2.
In another illustrative embodiment, described herein is a compound of the formula
wherein P is a phosphorus containing group; X is a leaving group; Q is a polyvalent linker, including but not limited to alkylene, heteroalkylene, or poly(oxyalkylene) each of which is optionally substituted; and n is an integer in the range from 1 to about 20.