Use of nucleic acids containing unmethylated CpG...

Details Use of nucleic acids containing unmethylated CpG... Use of nucleic acids containing unmethylated CpG...

1999-06-03

2002-06-18

Salimi, Ali R. (Department: 1648)

Drug, bio-affecting and body treating compositions

Nonspecific immunoeffector, per se ; or nonspecific...

C424S279100, C424S282100, C424S204100, C536S023720, C930S200000, C930S210000, C930S220000

Reexamination Certificate

active

06406705

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to adjuvants, and in particular to methods and products utilizing a synergistic combination of oligonucleotides having at least one unmethylated CpG dinucleotide (CpG ODN) and a non-nucleic acid adjuvant.
BACKGROUND OF THE INVENTION
Bacterial DNA, but not vertebrate DNA, has direct immunostimulatory effects on peripheral blood mononuclear cells (PBMC) in vitro (Krieg et al., 1995). This lymphocyte activation is due to unmethylated CpG dinucleotides, which are present at the expected frequency in bacterial DNA ({fraction (1/16)}), but are under-represented (CpG suppression, {fraction (1/50)} to {fraction (1/60)}) and methylated in vertebrate DNA. Activation may also be triggered by addition of synthetic oligodeoxynucleotides (ODN) that contain an unmethylated CpG dinucleotide in a particular sequence context. It appears likely that the rapid immune activation in response to CpG DNA may have evolved as one component of the innate immune defense mechanisms that recognize structural patterns specific to microbial molecules.
CpG DNA induces proliferation of almost all (>95%) B cells and increases immunoglobulin (Ig) secretion. This B cell activation by CpG DNA is T cell independent and antigen non-specific. However, B cell activation by low concentrations of CpG DNA has strong synergy with signals delivered through the B cell antigen receptor for both B cell proliferation and Ig secretion (Krieg et al., 1995). This strong synergy between the B cell signaling pathways triggered through the B cell antigen receptor and by CpG DNA promotes antigen specific immune responses. In addition to its direct effects on B cells, CpG DNA also directly activates monocytes, macrophages, and dendritic cells to secrete a variety of cytokines, including high levels of IL-12 (Klinman et al., 1996; Halpern et al., 1996; Cowdery et al., 1996). These cytokines stimulate natural killer (NK) cells to secrete gamma-interferon (IFN-&ggr;-) and have increased lytic activity (Klinman et al., 1996, supra; Cowdery et al., 1996, supra; Yamamoto et al., 1992; Ballas et al., 1996). Overall, CpG DNA induces a Th1 like pattern of cytokine production dominated by IL-12 and IFN-&ggr; with little secretion of Th2 cytokines (Klinman et al., 1996).
Hepatitis B virus (HBV) poses a serious world-wide health problem. The current HBV vaccines are subunit vaccines containing particles of HBV envelope protein(s) which include several B and T cell epitopes known collectively as HBV surface antigen (HBsAg). The HBsAg particles may be purified from the plasma of chronically infected individuals or more commonly are produced as recombinant proteins. These vaccines induce antibodies against HBsAg (anti-HBs), which confer protection if present in titers of at least 10 milli-International Units per milliliter (mIU/ml) (Ellis, 1993). The current subunit vaccines whch contain alum (a Th2 adjuvant), are safe and generally efficacious. They, however, fail to meet all current vaccination needs. For example, early vaccination of infants born to chronically infected mothers, as well as others in endemic areas, drastically reduces the rate of infection, but a significant proportion of these babies will still become chronically infected themselves (Lee et al., 1989; Chen et al., 1996). This could possibly be reduced if high titers of anti-HBs antibodies could be induced earlier and if there were HBV-specific CTL. In addition, there are certain individuals who fail to respond (non-responders) or do not attain protective levels of immunity (hypo-responders). Finally, there is an urgent need for an effective treatment for the estimated 350 million chronic carriers of HBV and a therapeutic vaccine could meet this need.
SUMMARY OF THE INVENTION
The present invention relates to methods and products for inducing an immune response. The invention is useful in one aspect as a method of inducing an antigen specific immune response in a subject. The method includes the steps of administering to the subject in order to induce an antigen specific immune response an antigen and a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a synergistic adjuvant response. In one embodiment the subject is an infant.
The CpG oligonucleotide and the non-nucleic acid adjuvant may be administered with any or all of the administrations of antigen. For instance the combination of adjuvants may be administered with a priming dose of antigen. In another embodiment the combination of adjuvants is administered with a boost dose of antigen. In some embodiments the subject is administered a priming dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide before the boost dose. In yet other embodiments the subject is administered a boost dose of antigen and oligonucleotide containing at least one unmethylated CpG dinucleotide after the priming dose.
The antigen may be any type of antigen known in the art. For example, the antigen may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids. Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
According to another aspect of the invention a method of inducing a Th1 immune response in a subject is provided. The method includes the step of administering to the subject in order to induce a Th1 immune response a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant, and wherein the combination of adjuvants is administered in an effective amount for inducing a Th1 immune response. In one embodiment the combination of adjuvants is administered simultaneously. In another embodiment the combination of adjuvants is administered sequentially. In some embodiments the combination of adjuvants is administered in an effective amount for inducing a synergistic Th1 immune response. In another aspect, the same method is performed but the subject is an infant and the Th1 response can be induced using CpG DNA alone, or CpG DNA in combination with a non-nucleic acid adjuvant at the same or different site, at the same or different time.
The invention in other aspects is a composition of a synergistic combination of adjuvants. The composition includes an effective amount for inducing a synergistic adjuvant response of a combination of adjuvants, wherein the combination of adjuvants includes at least one oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant. The composition may also include at least one antigen, which may be selected from the group consisting of peptides, polypeptides, cells, cell extracts, polysaccharides, polysaccharide conjugates, lipids, glycolipids and carbohydrates. Antigens may be given in a crude, purified or recombinant form and polypeptide/peptide antigens, including peptide mimics of polysaccharides, may also be encoded within nucleic acids. Antigens may be derived from an infectious pathogen such as a virus, bacterium, fungus or parasite, or the antigen may be a tumor antigen, or the antigen may be an allergen.
According to other aspects the invention includes a method for immunizing an infant. The method involves the step of administering to an infant an antigen and an oligonucleotide containing at least one unmethylated CpG dinucleotide and at least one non-nucleic acid adjuvant in a

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