JP2009132737A - Nucleic acid composition for stimulating immune response - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid comprising immunostimulation higher than that of a conventional nucleic acid. <P>SOLUTION: An immunostimulatory nucleic acid comprising CpG motifs, and a method of use thereof in immunostimulation is provided. The present invention is partly based on the surprising discovery of a new family of a nucleic acid inducing higher level immunostimulation than that of a conventional nucleic acid. The knowledge is surprising as one of reasons that more than 100 numbers of nucleic acid sequences had been screened before the nucleic acid sequence described in the specification was found. Immunostimulation effect of bacteria DNA can be simulated with a synthetic oligodeoxynucleotide (ODN) containing the CpG motifs. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

(Field of Invention)
The present invention relates generally to immunostimulatory nucleic acids, compositions thereof, and methods of using the immunostimulatory nucleic acids.

(Background of the Invention)
Bacterial DNA has an immunostimulatory effect to activate B cells and natural killer cells, whereas vertebrate DNA has no such effect (Tokunaga, T. et al., 1988. Jpn. J. Cancer Res. 79: 682-686; Tokunaga, T. et al., 1984, JCI 72: 955-962; Messina, JP et al., 19991, J. Immunol. C. Stein and AM Krieg, (Eds.), John Wiley and Sons, Inc., New York, NY, pp. 431-448). Currently, these immunostimulatory effects of bacterial DNA are present in unmethylated CpG dinucleotides, particularly in bacterial DNA, where there is a common base pair content (CpG motif), but in vertebrate DNA it is methylated and not It is understood that there are sufficient CpG dinucleotides (Non-Patent Document 1; Non-Patent Document 2).

By synthetic oligodeoxynucleotides (ODN) containing these CpG motifs,
It can mimic the immunostimulatory effect of bacterial DNA. Such CpG-ODN has a high stimulatory effect on human and mouse leukocytes, including B cell proliferation, cytokine and immunoglobulin secretion, natural killer (NK) cytolytic activity and IFN. -Γ secretion, and activation of dendritic cells (DCs) and other antigen presenting cells, including costimulatory molecules and Thl-like that is important in promoting the expression of cytokines (especially Thl-like cell responses) Secretes cytokines).

  The immunostimulatory effect of natural phosphodiester skeleton CpG-ODN is that the effect is essentially eliminated when methylation of CpG motif, change to GpC, or removal or modification of CpG motif otherwise. CpG specificity is high (Krieg et al., 1995 Nature 374: 546-549; Hartmann et al., 1999 Proc. Natl. Acad. Sci USA 96: 9305-10). Phosphate diester CpG-ODN can be formulated with lipids, alum, or other vehicles with reservoir properties or improved cellular uptake to enhance immune effects (Yamamoto et al., 1994 Microbiol. Immunol. 38: 831-836). Gramzinski et al., 1998 Mol. Med. 4: 109-118).

  In early studies it was thought that the immunostimulatory CpG motif follows the formula; purine-purine-CpG-pyrimidine-pyrimidine (Krieg et al., 1995 Nature 374: 546-549; Pisetsky, 1996 J. Immunol. 156: 421-). 423; Hacker et al., 1998 EMBO J. 17: 6230-6240; Lipford et al., 1998 Trends in Microbiol. 6: 497-500). However, mouse lymphocytes react fairly well to phosphodiester CpG that does not follow this “formula” (Yi et al., 1998 J. Inunol. 160: 5898-5906), and the same is true for humans. It is now revealed that this also applies to B cells and dendritic cells (Hartmann et al., 1999 Proc. Natl. Acad. Sci USA 96: 9305-10; Liang, 1996 J. Clin. Invest. 98: 1119-1129). Yes.

Several previous investigators have examined whether the nucleotide content of ODN has an effect independent of the ODN sequence. Interestingly, when an antisense ODN usually has a high content of GG, CCC, CC, CAC, and CG sequences, while compared to those expected to use random bases, The finding that the frequency of TT or TCC nucleotide sequences is reduced has been obtained (Smetsers et al., 1996 Antisense Nucleic Drug Development. 6: 63-67). This may be that the over-represented sequence contains a preferred target element for the antisense oligonucleotide and vice versa. One reason for avoiding the use of thymidine-rich ODN for antisense experiments is that free thymidines compete with ³ H-thymidine, which is often used in experiments where cell growth is evaluated by degradation of ODN by nucleases present in the cells. ((Matson et al., 1992 Antisense Research and Development 2: 325-330).).

Krieg et al., 1995 Nature 374: 546-549. Krieg, 1999 Biochim. Biophys. Acta 93321: 1-10

(Summary of the Invention)
The present invention is based in part on the surprising discovery of a new family of nucleic acids that induce higher levels of immune stimulation than known nucleic acids. This finding is surprising, partly because more than 100 nucleic acid sequences were screened prior to the discovery of the nucleic acid sequences disclosed herein.
One embodiment of the present invention is SEQ ID NO: 1 (ODN 10102) and SEQ ID NO: 19 (ODN 10).
103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105)
Or an immunostimulatory nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 141 (ODN 10106).

Another aspect of the invention is a method of stimulating an immune response in a subject in need of an immune response, wherein the amount is SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (
ODN 10105), or an immunostimulatory nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 141 (ODN 10106) is provided to a subject.

Various embodiments of the invention apply equally to the aspects described herein, some of which are described below.

In one embodiment, the immunostimulatory nucleic acid molecule comprises SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (
ODN 10105) or the nucleotide sequence of SEQ ID NO: 141 (ODN 10106).

In another embodiment, the composition further comprises an antigen. Alternatively, the subject to be treated is further administered an antigen. The antigen can be selected from the group consisting of a microbial antigen, a self antigen, a cancer antigen, and an allergen. However, it is not limited to that. In one embodiment, the microbial antigen is selected from the group consisting of a bacterial antigen, a viral antigen, a fungal antigen, and a parasitic antigen. In another embodiment, the antigen is encoded by a nucleic acid vector. In related embodiments, the nucleic acid vector is separate from the immunostimulatory nucleic acid. The antigen may be a peptide antigen.

In another embodiment, the composition further comprises an adjuvant, or the subject is further administered an adjuvant. The adjuvant may be a mucosal adjuvant, but is not limited thereto.

In another embodiment, the composition further comprises a cytokine or the subject is further administered a cytokine.

In yet another embodiment, the composition further comprises a therapeutic agent selected from the group consisting of an antibacterial agent, an anticancer agent, an allergy / asthma agent, or the subject further comprises a therapeutic agent selected from the same group. Be administered. In a related embodiment, the antimicrobial agent is selected from the group consisting of antimicrobial agents, antiviral agents, antifungal agents, and antiparasitic agents. In another embodiment, the anticancer agent is selected from the group consisting of chemotherapeutic agents, cancer vaccines, and immunotherapeutic agents. In yet another related embodiment, the allergy / asthma drug is a PDE-4 inhibitor, bronchodilator (bron
selector / β2 agonist, K + channel opener (channel)
opener), VLA-4 antagonist, neurocin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenase inhibitor, thromboxin (thro)
mboxin) A2 receptor antagonist, thromboxane
e) A2 antagonist, 5-lipox activating protein (5-lipox acti)
selected from the group consisting of inhibitors of proteases and protease inhibitors.

In some embodiments, the immunostimulatory nucleic acid can have a nucleotide backbone that includes at least one backbone modification. In one embodiment, the backbone modification is a phosphorothioate modification. In another embodiment, the nucleotide backbone is chimeric. In one embodiment,
The nucleotide backbone is completely modified.

  In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

In one embodiment, the immunostimulatory nucleic acid does not comprise a methylated CpG dinucleotide. In another embodiment, the immunostimulatory nucleic acid comprises at least 4 CpG motifs. In yet another example, the immunostimulatory nucleic acid is rich in T. In related embodiments, the immunostimulatory nucleic acid comprises a poly T sequence. In another embodiment, the immunostimulatory nucleic acid comprises a poly G sequence.

In certain embodiments, immunostimulatory nucleic acids are formulated in a variety of ways. In one embodiment, the immunostimulatory nucleic acid is formulated for oral administration. The immunostimulatory nucleic acid may be formulated as a nutritional supplement. In related embodiments, the nutritional supplement is formulated as a capsule, a pill, or a sublingual tablet. In another embodiment,
An immune activator is formulated for topical administration. The immunostimulatory nucleic acid may be formulated for parenteral administration, or may be formulated in a sustained release device. The sustained-release device can be made into fine particles, but is not limited thereto. In another embodiment, the immunostimulatory nucleic acid is formulated for delivery to the mucosal surface. The mucosal surface can be selected from the group consisting of the oral surface, nasal surface, vaginal surface, and ocular surface, but is not limited thereto.

In one embodiment, the immunostimulatory nucleic acid stimulates a mucosal immune response. In other embodiments, the immunostimulatory nucleic acid stimulates a systemic immune response. In important embodiments, the immunostimulatory nucleic acid stimulates both mucosal and systemic immune responses. In some embodiments, the immune response is an antigen-specific immune response. In related embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate a mucosal immune response. In other embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate a systemic immune response. In yet other embodiments, the immunostimulatory nucleic acid is provided in an amount effective to stimulate a natural immune response.

In many embodiments, the immunostimulatory nucleic acid is intended for the treatment or prevention of various diseases. Thus, in one embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent an infection. In another embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent allergy. In yet another example, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent asthma. In yet another embodiment, the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent cancer.

In a related embodiment, the infection is a herpes simplex virus infection. In another embodiment, the immunostimulatory nucleic acid is intended for administration to a subject suffering from or at risk of developing an infection. The infection can be selected from the group consisting of a bacterial infection, a viral infection, a fungal infection, and a parasitic infection. In one embodiment,
Viral infections include human immunodeficiency virus (HIV-1 and HIV-2), human type I T lymphoproliferative virus (HTLV-I), human type II T lymphoproliferative virus (HTLV-I).
I), type I herpes simplex virus (HSV-1), type 2 herpes simplex virus (HSV-
2), human papilloma virus (multiple), hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis D virus, Epstein-Barr virus (EBV)
), Cytomegalovirus, and Molluscum infectious virus. In important embodiments, the viral infection is a herpes simplex virus infection.

In another embodiment, the infectious disease is a microbial species selected from the group consisting of herpesviridae, retroviridae, orthomyxoviridae, toxoplasma, hemophilus, campylobacter, clostridium, E. coli, and staphylococci It is an infection caused by. In related embodiments, the antigen or antigen contained in the composition administered to the subject is derived from one of the aforementioned species.

  In certain embodiments, the infection is a SARS infection or a sarpox infection.

In another embodiment, the immunostimulatory nucleic acid is a subject that is allergic or at risk of developing allergy, a subject that presents or is at risk of developing asthma, or suffers from cancer. For administration to a subject who is or is at risk of developing cancer.

In an embodiment relating to treatment to a subject, the method further comprises isolating immune cells from the subject, and an amount of the immune activating nucleic acid effective to activate the immune cells and the immune cells. It may further comprise contacting and administering the activated immune cells again to the subject. In one embodiment, the immune cell is a white blood cell. In another embodiment, the immune cell is a dendritic cell. In another embodiment, the method comprises contacting the immune cell with an antigen.
In addition.

In important embodiments, the subject is a human. In another embodiment, the subject is a dog, cat,
Selected from the group consisting of horses, cows, pigs, sheep, goats, birds, monkeys, and fish.

Accordingly, the methods provided herein can be used on a subject suffering from or at risk of developing an infection, the method treating an infection in a subject. Or a method for prevention. In addition, this method can be used for a subject suffering from asthma or a subject at risk of developing asthma, and the method is a method for treating or preventing asthma. This method can be used on a subject suffering from allergy or at risk of developing an allergy, the method being a method for treating or preventing allergy. Furthermore, the method can be used for a subject suffering from cancer or a subject at risk of developing cancer, the method being a method for treating or preventing cancer. In one embodiment, the cancer is biliary tract cancer, bone cancer, brain and central nervous system (C
NS) cancer, breast cancer, cervical cancer, choriocarcinoma, colon cancer, connective tissue cancer, endometrial cancer, esophageal cancer, eye cancer, stomach cancer, Hodgkin lymphoma, carcinoma in situ, laryngeal cancer, lymphoma, liver cancer, Consists of lung cancer (eg small and non-small cells), melanoma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and kidney cancer Selected from the group.

In yet another embodiment of the methods for treatment or prevention provided herein, the method further comprises administering an antibody specific for a cell surface antigen, wherein the immune response is antigen dependent. Causes cytotoxic activity (ADCC).

In another aspect of the invention, a method of preventing disease in a subject includes preventing the subject's disease by periodically administering an immunostimulatory nucleic acid to the subject. The activated nucleic acid is SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (ODN 10105) or SEQ ID NO: 1.
It has a nucleotide sequence comprising 41 (ODN 10106).

In yet another aspect, the present invention provides a method of inducing an innate immune response, wherein the method comprises an amount of an immunostimulatory nucleic acid effective to activate the innate immune response in a subject. The immunostimulatory nucleic acid comprises SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19
(ODN 10103), SEQ ID NO: 45 (ODN 10104), SEQ ID NO: 118 (OD
N 10105) or SEQ ID NO: 141 (ODN 10106).

In yet another aspect, the invention provides a method of identifying an immunostimulatory nucleic acid comprising: SEQ ID NO: 1 (ODN 10102), SEQ ID NO: 19 (ODN 10103), SEQ ID NO: 45 (ODN 10104) Measuring an activation control level of an immune cell population contacted with an immunostimulatory nucleic acid comprising the nucleotide sequence of SEQ ID NO: 118 (ODN 10105) or SEQ ID NO: 141 (ODN 10106), and immunization contacted with the test nucleic acid Measuring an activation test level of a cell population and comparing an activation control level with an activation test level, wherein a test level equal to or greater than the control level is an immune activation Indicates nucleic acid.
The present invention further provides the following items.
(Item 1)
A composition comprising an immunostimulatory nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.
(Item 2)
142. The composition of item 1, wherein the immunostimulatory nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.
(Item 3)
Item 2. The composition according to Item 1, further comprising an antigen.
(Item 4)
Item 4. The composition according to Item 3, wherein the antigen is selected from the group consisting of a microbial antigen, a cancer antigen, and an allergen.
(Item 5)
Item 5. The composition according to Item 4, wherein the microbial antigen is selected from the group consisting of a microbial antigen, a viral antigen, a fungal antigen, and a parasitic antigen.
(Item 6)
4. The composition of item 3, wherein the antigen is encoded by a nucleic acid vector.
(Item 7)
4. The composition according to item 3, wherein the nucleic acid vector is different from the immunostimulatory nucleic acid.
(Item 8)
4. The composition according to item 3, wherein the antigen is a peptide antigen.
(Item 9)
The composition according to item 1, further comprising an adjuvant.
(Item 10)
Item 10. The composition according to Item 9, wherein the adjuvant is a mucosal adjuvant.
(Item 11)
Item 2. The composition according to Item 1, further comprising a cytokine.
(Item 12)
The composition according to item 1, further comprising a therapeutic agent selected from the group consisting of an anti-microbiological agent, an anticancer agent, and an allergy / asthma medicine.
(Item 13)
13. A composition according to item 12, wherein the antibacterial agent is selected from the group consisting of an anti-bacterial agent, an antiviral agent, an antifungal agent, and an antiparasitic agent.
(Item 14)
13. The composition according to item 12, wherein the anticancer agent is selected from the group consisting of a chemotherapeutic agent, a cancer vaccine, and an immunotherapeutic agent.
(Item 15)
The allergy / asthma medicament is a PDE-4 inhibitor, a bronchodilator / β-2 agonist, a K + channel opener, a VLA-4 antagonist, a neurocin antagonist, a TXA2 synthesis inhibitor, xanthanine ( xanthane), arachidonic acid antagonists, 5 lipoxygenase inhibitors, thromboxin A2 receptor antagonists, thromboxane A2 antagonists, inhibitors of 5-lipox activation protein, and protease inhibitors Item 12 selected from Of the composition.
(Item 17)
The composition according to item 1, wherein the immunostimulatory nucleic acid has a nucleotide backbone comprising at least one backbone modification.
(Item 18)
18. A composition according to item 17, wherein the backbone modification is a phosphorothioate modification.
(Item 19)
18. A composition according to item 17, wherein the nucleotide backbone is chimeric.
(Item 20)
18. A composition according to item 17, wherein the nucleotide backbone is fully modified.
(Item 21)
The composition of item 1, further comprising a pharmaceutically acceptable carrier.
(Item 22)
Item 2. The composition according to Item 1, wherein the immunostimulatory nucleic acid does not comprise a methylated CpG dinucleotide.
(Item 23)
The composition of item 1, wherein the immunostimulatory nucleic acid comprises at least four CpG moieties.
(Item 24)
Item 2. The composition according to Item 1, wherein the immunostimulatory nucleic acid is T-rich.
(Item 25)
The composition of item 1, wherein the immunostimulatory nucleic acid comprises a poly-T sequence.
(Item 26)
The composition of item 1, wherein the immunostimulatory nucleic acid comprises a poly G sequence.
(Item 27)
The composition of item 1, wherein the immunostimulatory nucleic acid is formulated for oral administration.
(Item 28)
The composition of item 1, wherein the immunostimulatory nucleic acid is formulated as a nutritional supplement.
(Item 29)
29. A composition according to item 28, wherein the immune adjuvant is formulated as a capsule, tablet or sublingual tablet.
(Item 30)
The composition of item 1, wherein the immunostimulatory nucleic acid is formulated for topical administration.
(Item 31)
Item 2. The composition of item 1, wherein the immunostimulatory nucleic acid is formulated for parenteral administration.
(Item 32)
The composition of item 1, wherein the immunostimulatory nucleic acid is released in a sustained release device.
(Item 33)
The composition of item 1, wherein the immunostimulatory nucleic acid is formulated to be delivered to a mucosal surface.
(Item 34)
Item 2. The composition according to Item 1, wherein the mucosal surface is selected from the group consisting of the oral cavity, nose, rectum, vagina, and ocular surface.
(Item 35)
The composition of item 1, wherein the immunostimulatory nucleic acid stimulates a mucosal immune response.
(Item 36)
The composition of item 1, wherein the immunostimulatory nucleic acid stimulates a systemic immune response.
(Item 37)
The composition of item 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate a mucosal immune response.
(Item 38)
The composition of claim 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate a systemic immune response.
(Item 39)
The composition of item 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to stimulate an innate immune response.
(Item 40)
The composition of item 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent an infectious disease.
(Item 41)
The composition of item 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent allergy.
(Item 42)
The composition of item 1, wherein the immunostimulatory nucleic acid is provided in an amount effective to treat or prevent asthma.
(Item 43)
The composition of item 1, wherein said immunostimulatory nucleic acid is provided in an amount effective to treat or prevent cancer.
(Item 44)
33. The composition according to item 32, wherein the sustained-release device is a fine particle.
(Item 45)
41. A composition according to item 40, wherein the infectious disease is a herpes simplex virus infection.
(Item 46)
A method of stimulating an immune response in a subject in need of stimulating an immune response, said method being an amount effective to stimulate an immune response, SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45. A method comprising administering to a subject an immunostimulatory nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 118 or SEQ ID NO: 141.
(Item 47)
49. The method of item 46, wherein the subject has an infection or is at risk of developing an infection.
(Item 48)
48. The method of item 47, wherein the infection is selected from the group consisting of a bacterial infection, a viral infection, a fungal infection and a parasitic infection.
(Item 49)
Said viral infection is human immunodeficiency virus (HIV-1 and HIV-2), type I human T lymphotrophic virus (HTLV-I), type II human T lymphotrophic virus (HTLV-II), type I herpes simplex virus (HSV-1), type II herpes simplex virus (HSV-2), human papillomavirus (multiple), hepatitis A virus, hepatitis B virus, hepatitis C virus and hepatitis D virus, EB virus (EBV), 49. A method according to item 48, wherein the method is selected from the group consisting of cytomegalovirus and infectious molluscumoma virus.
.
(Item 50)
50. The method of item 49, wherein the viral infection is a herpes simplex virus infection.
(Item 51)
49. The method of item 46, wherein the subject has an allergy or is at risk of developing an allergy.
(Item 52)
49. The method of item 46, wherein the subject has asthma or is at risk of developing asthma.
(Item 53)
49. The method of item 46, wherein the subject has cancer or is at risk of developing cancer.
(Item 54)
47. The method of item 46, further comprising administering an antigen to the subject.
(Item 55)
54. The method of item 53, wherein the antigen is selected from the group consisting of a microbial antigen, a cancer antigen, a self antigen and an allergen.
(Item 56)
55. The method of item 54, wherein the microbial antigen is selected from the group consisting of a bacterial antigen, a viral antigen, a fungal antigen and a parasitic antigen.
(Item 57)
The antigen is a herpesviridae, retroviridae, orthomyxofamily, toxoplasma, hemophilus, campylobacter, clostridium, E. coli. 56. The method of item 55, wherein the method is derived from a microorganism selected from the group consisting of E. coli and staphylococci.
(Item 58)
47. A method according to item 46, wherein the immune response is an antigen-specific immune response.
(Item 59)
54. The method of item 53, wherein the antigen is encoded by a nucleic acid vector.
(Item 60)
60. The method of item 59, wherein the nucleic acid vector is separate from the immunostimulatory nucleic acid.
(Item 61)
55. A method according to item 54, wherein the antigen is a peptide antigen.
(Item 62)
49. The method of item 46, further comprising administering an adjuvant to the subject.
(Item 63)
63. The method of item 62, wherein the adjuvant is a mucosal adjuvant.
(Item 64)
47. The method of item 46, further comprising administering a second therapeutic agent to the subject.
(Item 65)
65. A method according to item 64, wherein the second therapeutic agent is an antibacterial agent.
(Item 66)
66. The method of item 65, wherein the antibacterial agent is selected from the group consisting of an antibacterial agent, an antiviral agent, an antifungal agent, and an antiparasitic agent.
(Item 67)
65. The method of item 64, wherein the second therapeutic agent is an anticancer agent.
(Item 68)
68. The method of item 67, wherein the anticancer agent is selected from the group consisting of a chemotherapeutic agent, a cancer vaccine, and an immunotherapeutic agent.
(Item 69)
65. The method of item 64, wherein the second therapeutic agent is an allergy / asthma medicine.
(Item 70)
The allergy / asthma medicine is a PDE-4 inhibitor, bronchodilator / β-2 agonist, K + channel opener, VLA-4 antagonist, neurosin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenase inhibitor, thrombosin A2 70. The method of item 69, selected from the group consisting of a receptor antagonist, a thromboxane A2 antagonist, an inhibitor of 5-lipox activating protein, and a protease inhibitor.
(Item 71)
47. The method of item 46, wherein said immunostimulatory nucleic acid has a nucleotide backbone comprising at least one backbone modification.
(Item 72)
72. A method according to item 71, wherein the backbone modification is phosphorothioate modification.
(Item 73)
72. A method according to item 71, wherein the nucleotide backbone is chimera.
(Item 74)
72. A method according to item 71, wherein the nucleotide backbone is completely modified.
(Item 75)
49. The method of item 46, wherein the immunostimulatory nucleic acid does not comprise a methylated CpG dinucleotide.
(Item 76)
47. The method of item 46, wherein the immunostimulatory nucleic acid comprises a poly G sequence.
(Item 77)
49. The method of item 46, wherein the immunostimulatory nucleic acid is administered orally.
(Item 78)
49. The method of item 46, wherein the immunostimulatory nucleic acid is administered locally.
(Item 79)
49. The method of item 46, wherein the immunostimulatory nucleic acid is administered parenterally.
(Item 80)
49. The method of item 46, wherein the immunostimulatory nucleic acid is administered in a sustained release device.
(Item 81)
49. The method of item 46, wherein the immunostimulatory nucleic acid is administered to the mucosal surface.
(Item 82)
47. A method according to item 46, wherein the immune response is a mucosal immune response.
(Item 83)
47. A method according to item 46, wherein the immune response is a systemic immune response.
(Item 84)
82. The method of item 81, wherein the mucosal surface is selected from the group consisting of an oral cavity, nose, rectum, vagina, and eye surface.
(Item 85)
47. A method according to item 46, the method comprising:
Simply obtaining immune cells from the subject,
Contacting the immune cells with an amount of the immunostimulatory nucleic acid effective to activate the immune cells; and
Re-administering the activated immune cells to the subject;
Including the method.
(Item 86)
86. The method of item 85, wherein the immune cell is a leukocyte.
(Item 87)
86. The method of item 85, wherein the immune cell is a dendritic cell.
(Item 88)
86. The method according to item 85, further comprising contacting the immune cell with the antigen.
(Item 89)
47. The method of item 46, wherein the subject is a human.
(Item 90)
49. The method of item 46, wherein the subject is selected from the group consisting of dogs, cats, horses, pigs, sheep, goats, birds, monkeys, and fish.
(Item 91)
47. Item 46, wherein the subject has or is at risk of developing an infectious disease, wherein the method is a method of treating or preventing the infectious disease. Method.
(Item 92)
49. The method of item 46, wherein the subject has or is at risk of developing asthma, wherein the method is a method of treating or preventing the asthma in the subject.
(Item 93)
49. The method of item 46, wherein the subject has an allergy or is at risk of developing an allergy, wherein the method is a method of treating or preventing the allergy.
(Item 94)
49. The method of item 46, wherein the subject has cancer or is at risk of developing cancer, wherein the method is a method of treating or preventing the cancer.
(Item 95)
95. The method of item 94, wherein the cancer is biliary tract cancer; bone cancer; brain and central nervous system (CNS) cancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer; Malignant cancer; Esophageal cancer; Eye cancer; Gastric cancer; Hodgkin lymphoma; Intraepithelial neoplasm; Laryngeal cancer; Lymphoma; Liver cancer; A method selected from the group consisting of: ovarian cancer; pancreatic cancer; prostate cancer; rectal cancer; sarcoma; skin cancer; testicular cancer;
(Item 96)
49. The method of item 46, further comprising administering an antibody specific for a cell surface antigen, wherein the immune response is antigen dependent cellular cytotoxicity (ADCC). ) How to cause.
(Item 97)
A method of preventing a disease in a subject comprising administering an immunostimulatory nucleic acid to the subject in a standard basis for preventing the disease in the subject. Wherein the immunostimulatory nucleic acid has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.
(Item 98)
A method of inducing an innate immune response comprising administering to the subject an immunostimulatory nucleic acid in an amount effective to stimulate the innate immune response, wherein: The method wherein the immunostimulatory nucleic acid has the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 or SEQ ID NO: 141.
(Item 99)
A method of identifying an immunostimulatory nucleic acid comprising:
Measuring a control level of activation of an immune cell population contacted with an immunostimulatory nucleic acid, the immunostimulatory nucleic acid comprising SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118 141 comprising a nucleotide sequence of 141,
Measuring a test level of activation of the contacted immune cell population in contact with the test nucleic acid;
and
Comparing the activation control level with the activation test level;
Wherein a test level that is greater than or equal to the control level is an indicator of an immunostimulatory nucleic acid.
These and other aspects and embodiments of the invention are described in further detail herein.

TLR9 engagement with ODN 7909 and 10102. As described in Example 1, TLR9 expressing cultured cell lines were incubated with the indicated concentrations of ODN. The average stimulation index on the medium control is illustrated. IL-1 was used as a positive control for the reporter gene. Incubation of PBMC with CpG ODN upregulates the activity marker CD86 by B cells. Human PBMC were incubated with ODN 7909 and 10102 for 48 hours at the indicated concentrations. Shown is the average percentage of CD86-expressing CD19 positive B cells (measured by flow cytometry) from three different donors. B cell proliferation induced by CpG ODN 7909 and 10102. PBMC pre-incubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentrations. Cells were harvested and the reduction of the CFSE strain on proliferating CD19 positive B cells was measured by flow cytometry on three different donors (see also Example 1). INFα secretion induced by ODN 7909 and 10102. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 1). For each concentration, the average, minimum, and maximum amounts of INFα obtained from three different donors are illustrated. IP-10 secretion induced by ODN 7909 and 10102. Three different donor human PBMCs were incubated with the indicated concentrations of ODN for 48 hours. The supernatant was collected and IP-10 was measured by ELISA (see Example 1). For each concentration, the average, minimum, and maximum amounts of IP-10 obtained from three different donors are illustrated. IL-10 secretion induced by ODN 7909 and 10102. Triplicate blood donor PBMCs were incubated with the indicated concentrations of ODN 7907, 10102 or control ODN. The supernatant was collected and IL-10 was measured by ELISA. For each concentration, the average, minimum, and maximum amounts of I-10 obtained from three different donors are illustrated. TNFα secretion in response to ODN 7909 and 10102. Three different blood donor PBMCs were incubated for 24 hours with the indicated concentrations of ODN 7907, 10102 or control ODN. The supernatant was collected and TNFα was measured by ELISA. For each concentration, the average, minimum, and maximum amounts from three different donors are illustrated. Untreated BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODN 10102 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity measured. Each bar represents the stimulation index (counts of cells incubated / minute (CPM) / CMP of cells incubated in medium). Untreated BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909, 10102, or control ODN2137. Supernatants were collected at 6 hours (TNFα, panel D), 24 hours (IL-12, panel B), or 48 hours (IL-6, panel C, and IL-10, panel A). Untreated BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909, 10102. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 10102, 7909, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for total IgG levels against HBsAg (anti-HBs). Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the entire group (n = 10). The titer was defined as the highest dilution at which the absorbance was twice the absorbance of non-immune plasma with a category value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 7909, 10102 or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for IgG1 and IgG2a levels (antibody HBs) against HBsAg. Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the entire ELISA weekend dilution group (n = 10). The titer was defined as the highest dilution at which the absorbance was twice the absorbance of non-immune plasma with a category value of 0.05. TLR9 engagement with ODN 7909 and 10103. TLR-9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 2. For four independent experiments, the average stimulation index on the media control is illustrated. IL-1 was used as a positive control for the reporter gene. Incubation of PBMC with CpG ODN upregulates the activity marker CD86 by B cells. Human PBMC were incubated with ODN 7909 and 10103 and control ODN for 48 hours at the indicated concentrations. Shown is the average percentage of CD86-expressing CD19 positive B cells (measured by flow cytometry) from three different donors. B cell proliferation induced by CpG ODN 7909 and 10103. PBMC pre-incubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentrations. Cells were harvested and the decrease in the CFSE line relative to proliferating CD19 positive B cells was measured by flow cytometry (see also Example 2). INF-α secretion induced by ODN 7909 and 10103. Six different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 2). For each concentration, the amount of INFα obtained from 6 different donors is illustrated. IP-10 secretion induced by ODN 7909 and 10103. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Example 2). For each concentration, the average amount of IP-10 obtained from three different donors is illustrated. IL-10 secretion relative to incubation with different concentrations of 7909, 10103, and control ODN. The means from three different donors obtained by incubation over 48 hours is illustrated. TNF-α secretion: PBMCs from three different blood donors were incubated with the indicated concentrations of ODN 7909, 10103, or controls for 48 hours. The supernatant was collected and TNF-α was measured by ELISA. Average values for triplicate donors are shown. Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml or 2.5 × 10 ⁶ / m1) with medium (negative control) or different amounts of CpG ODN 7909 (white bar), 10103 (black bar) Incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity measured. Each bar represents the stimulation index (counts of cells incubated / minute (CPM) / CMP of cells incubated in medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909, 10103, or control ODN2137. Supernatants were collected at 6 hours (TNF-α, Panel D), 24 hours (IL-12, Panel B), or 48 hours (IL-6, Panel C, and IL-10, Panel A). It was. Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909 and 10103. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 10103, 7909, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for total IgG levels against HBsAg (anti-HBs). Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the entire group (n = 5). The titer was defined as the maximum dilution that produced an absorbance of twice the absorbance of non-immune plasma with a category value of 0.05. Adult (6-8 week) BALB / c mice were immunized with 1 mg HBsAg alone or in combination with 10 mg CpGODN 7909, 10103, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for IgG1 and IgG2a levels (antibody HBs) against HBsAg. Each bar represents the geometric mean (± SEM) for the entire group of ELISA end point dilution titers (n = 5). The titer was defined as the maximum dilution that produced an absorbance of twice the absorbance of non-immune plasma with a category value of 0.05. Adult (6-8 week) BALB / c mice were immunized with 1 mg HBsAg in combination with either CpG-ODN (10 μg) 7909 or 10103. At 4 weeks after immunization, the spleen was removed and the splenocytes were used for measurement of CTL activity by ⁵¹ Cr release assay. CTL activity was expressed as mean% specific cell lysis (± SEM) for groups of animals (n = 5) at different effector: target ratios. 2 is a graph showing mean pathological scores as a function of days after infection in mice challenged with HSV-2 and administered nucleic acid 10104. FIG. FIG. 4 is a graph showing survival as a function of days after infection in mice receiving antigen challenge with HSV-2 and receiving nucleic acid 10104. FIG. FIG. 10 is a bar graph showing human IFN-α induction in human PBMC after 48 hours by nucleic acid 10104 or control. IFN-α was measured by ELISA and the results are shown as mean ± SEM from triplicate blood donors. FIG. 10 is a bar graph showing human IL-10 induction in human PBMC after 48 hours by nucleic acid 10104 or control. IL-10 was measured by ELISA and the results are shown as mean ± SEM from triplicate blood donors. FIG. 10 is a bar graph showing human TLR9-mediated NFkB stimulation after 16 hours exposure to nucleic acid 10104 or control. Stimulation was measured using a reporter gene upstream assay. TLR9 engagement with ODN 7909 and 10105. TLR9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 4. For four independent experiments, the average stimulation index on the media control is illustrated. IL-1 was used as a positive control for the reporter gene. During incubation of PBMC with CpG-ODN, B cell upstream regulates the activity marker CD86. Human PBMCs were incubated with ODN 7909 and 10105 as for the control ODN at the indicated concentrations for 48 hours. The figure shows the average percentage of CD86-expressing CD19-positive B cells from three different donors (measured by flow cytometry). B cell proliferation induced by CpG ODN 7909 and 10105. PBMC preincubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentrations. Cells were harvested and the reduction of the CFSE line relative to proliferating CD19 positive B cells was measured by flow cytometry (see also Example 4). IFN-α secretion induced by ODN 7909 and 10105. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 4). The average amount of IFN-α obtained is plotted for three different donors at each concentration. IP-10 secretion induced by ODN 7909 and 10105. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Example 4). The average amount of IP-10 obtained is illustrated for three different donors at each concentration. Time kinetics of IFN-α secretion. Two different blood donor PBMCs were incubated with the indicated concentrations of ODN 7909, 10105 or control ODN for 8 or 24 hours. The supernatant was collected and IFN-α was measured by ELISA. The individual IFN-α amounts obtained at different times for the two donors are shown. Time kinetics of IFN-α secretion. Two different blood donor PBMCs were incubated with the indicated concentrations of ODN 7909, 10105, or controls for 36 or 48 hours. The supernatant was collected and IFN-α was measured by ELISA. The individual IFN-α amounts obtained at different time points are shown for the two donors. Time kinetics of IL-10 secretion. Three different blood donor PBMCs were incubated with the indicated concentrations of ODN 7909, 10105 or control ODN for 8, 24, or 48 hours. The supernatant was collected and IL-10 was measured by ELISA. The individual IL-10 amounts obtained at different time points for the three donors are shown. Time kinetics of IL-10 secretion. The same experiment as in Example 8 is shown. The average amount of IL-10 at each concentration and time point between triplicate donors was calculated. Naive BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODNs 7909 and 10105 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity measured. Each bar represents the stimulation index (counts of cells incubated / minute (CMP) / CMP of cells incubated in medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909, 10105, or control ODN2137. Supernatants were collected at 6 hours (TNF-α, Panel D), 24 hours (IL-12, Panel B), or 48 hours (IL-6, Panel C, and IL-10, Panel A). It was. Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909 and 10105. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 10105, 7909, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for total IgG levels against HBsAg (anti-HBs). Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the entire group (n = 10). The titer was defined as the maximum dilution that produced an absorbance of twice the absorbance of non-immune plasma with a compartment value of 0.05. Adult (6-8 week) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with 10 μg CpGODN 7909, 10105, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for IgG1 and IgG2a levels (antibody HBs) against HBsAg. Each bar represents the geometric mean (± SEM) for the entire group of ELISA end point dilution titers (n = 10). The titer was defined as the maximum dilution that gave an absorbance twice that of non-immune plasma with a compartment value of 0.05. B cell proliferation induced by CpGODN. PBMCs from normal and healthy subjects (n = 10) or subjects chronically infected with HCV (n = 10) at a concentration of 0.5 × 10 ⁶ / ml were added to medium (negative control) or Incubated with increasing amounts of CpGODN 7909 and 10106 or 4010 at 6 μg / mL. Cells were pulsed with ³ H-thymidine (1 μCi / well) for 16-18 hours after 5 days of incubation, harvested, and radioactivity measured. Each bar represents the average stimulation index (counts of cells incubated with ODN / min (CPM) / CPM of cells incubated with medium). Secretion of IFN-α induced by CpGODN. Human PBMC from normal and healthy subjects and subjects chronically infected with HCV were incubated with controls ODN 4010, 7909, or 10106 at concentrations in the range of 1-6 μg / mL. The supernatant was collected and IFN-α was measured by ELISA (see Example 5). The detection limit of this assay is 31.2 pg / mL, and subjects with IFN-α results below the detection limit are not represented on the graph. Average values shown as straight lines were determined by detectable IFN-α for these subjects. IP-10 secretion induced by CpGODN. Human PBMC from 10 normal and healthy subjects and 10 HCV chronically affected subjects were incubated with control ODN 4010, 7909, or 10106 at a concentration range of 1-6 μg / mL. The supernatant was collected and IP-10 was measured by ELISA (detection limit 15.6 pg / mL) (see Example 5). IL-10 secretion induced by CpGODN. Human PBMC from 10 normal and healthy subjects and 10 HCV chronically affected subjects were incubated with control ODN 4010, 7909, or 10106 at a concentration range of 1-6 μg / mL. The supernatant was collected and IL-10 was measured by ELISA (see Example 5). The detection limit for the ELISA assay is 23.4 pg / mL. If the treatment group has undetectable IL-10 concentrations, the number of subjects with detectable IL-10 is shown on the graph as a percentage of the total number of patients evaluated. The determined mean and standard deviation are for patients with detectable IL-10. TLR9 engagement with ODN 7909 and 10106. TLR9 expressing cell lines were incubated with the indicated concentrations of ODN as described in Example 5. The average stimulation index on the medium control is illustrated. IL-1 was used as a positive control for the reporter gene. B cells up-regulate the activity marker CD86 during incubation of PBMC with CpG-ODN. Human PBMC were incubated with ODN 7909 and 10106 for 48 hours at the indicated concentrations. Shown is the average percentage of CD86-expressing CD19 positive B cells (measured by flow cytometry) from three different donors. B cell proliferation induced by CpG-ODN 7909 and 10106. PBMC preincubated with the dye CFSE were cultured for 5 days in the presence or absence of the indicated ODN concentrations. Cells were harvested and the reduction of the CFSE line relative to proliferating CD19 positive B cells was measured by flow cytometry for three different donors (see also Example 5). INF-α secretion induced by ODN 7909 and 10106. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IFN-α was measured by ELISA (see Example 5). The average, minimum and maximum amounts of IFN-α obtained are plotted for three different donors at each concentration. IP-10 secretion induced by ODN 7909 and 10106. Three different donor human PBMCs were incubated for 48 hours with the indicated concentrations of ODN. The supernatant was collected and IP-10 was measured by ELISA (see Materials and Methods). The average, minimum, and maximum amounts of IP-10 obtained are illustrated for three different donors at each concentration. IL-10 secretion. Three different blood donor PBMCs were incubated with the indicated concentrations of ODN 7909, 10106 or controls. The supernatant was collected and IL-10 was measured by ELISA. The average, minimum, and maximum amounts of IL-10 obtained from three donors are illustrated. TNF-α secretion: PBMCs from three different blood donors were incubated for 16 hours with the indicated concentrations of ODN 7909, 10106 or controls. The supernatant was collected and TNF-α was measured by ELISA. The average, minimum and maximum amounts of triplicate donors are illustrated. Naive BALB / c mouse spleen cells (5 × ¹⁰ 6 / ml or 2.5 × ¹⁰ 6 / m1) of medium (negative control) or with different amounts of CpG-ODNs 7909 and 10106 and incubated. Cells were pulsed with ³ H thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity measured. Each bar represents the stimulation index (counts of cells incubated / minute (CPM) / CPM of cells incubated in medium). Naive BALB / c mouse splenocytes (5 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG-ODN 7909, 10106, or control ODN2137. Supernatants were collected at 6 hours (TNF-α, Panel D), 24 hours (IL-12, Panel B), or 48 hours (IL-6, Panel C, and IL-10, Panel A). It was. Naive BALB / c mouse splenocytes (30 × 10 ⁶ / ml) were incubated with medium (negative control) or different amounts of CpG ODN 7909 and 10106. NK activity was measured using a ⁵¹ Cr release assay. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with CpG-ODN (10 μg) 10106, 7909, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for total IgG levels against HBsAg (anti-HBs). Each bar represents the geometric mean (± SEM) of ELISA endpoint dilution titers for the entire group (n = 10). The titer was defined as the maximum dilution that produced an absorbance of twice the absorbance of non-immune plasma with a compartment value of 0.05. Adult (6-8 weeks) BALB / c mice were immunized with 1 μg HBsAg alone or in combination with 10 μg CpGODN 7909, 10106, or control ODN (10 μg) 2137. Animals were bled 4 weeks after immunization and plasma was assayed for IgG1 and IgG2a levels (antibody HBs) against HBsAg. Each bar represents the geometric mean (± SEM) for the entire group of ELISA end-point dilution titers (n = 10). The titer was defined as the maximum dilution that gave an absorbance twice that of non-immune plasma with a compartment value of 0.05. FIG. 61A shows the effect of local CpG delivery using a BEMA disc on the local pathology of mice after intravaginal challenge with HSV-2. FIG. 61B shows the effect of local CpG delivery with saline on the local pathology of mice after intravaginal challenge with HSV-2. FIG. 62A shows the effect of local CpG delivery using BEMA discs on survival of mice after intravaginal challenge with HSV-2. FIG. 62B shows the effect of local CpG delivery with saline on survival of mice following intravaginal challenge with HSV-2. Figure 3 shows the effect of parenteral CpG10104 delivery on IP-10 levels in mouse plasma. Figure 3 shows the effect of parenteral CpG10104 delivery on mouse plasma IFN-g levels. Figure 3 shows the effect of intravaginal CpG10104 delivery on mouse plasma IP-10 levels. Figure 3 shows the effect of local CpG delivery on mouse local pathology after intravaginal challenge with HSV-2. Figure 3 shows the effect of local CpG delivery on survival of mice after intravaginal challenge with HSV-2. Figure 6 shows the effect of intravaginal CpG10104 delivery on IP-10 levels of mouse vaginal lavage. FIG. 69A: shows the effect of local CpG delivery on mouse local pathology after intravaginal challenge with HSV-2. FIG. 69B shows the effect of local CpG delivery on survival of mice following intravaginal challenge with HSV-2. FIG. 70A shows that CpG10104 is as good as CpG7909 for increasing the humoral response of BALB / C mice to HBsAg in the absence of alum. FIG. 70B shows that CpG10104 is as good as CpG7909 for increasing the humoral response of BALB / C mice to HBsAg in the presence of alum. FIG. 71A shows that CpG10104 is as good as CpG7909 in promoting Th1-mediated immune response to HBsAg in BALB / C mice (as measured by high IgG2a compared to IgG1 titer) in the absence of alum. FIG. 71B shows that CpG10104 is as good as CpG7909 in promoting Th1-mediated immune response to HBsAg in BALB / C mice (as measured by high IgG2a compared to IgG1 titer) in the presence of alum.

(Detailed description of the invention)
CpG-containing nucleic acids can be used to stimulate the immune system, thereby helping to protect against infection after treatment of cancer, infections, allergies, asthma, and other disorders and cancer chemotherapy, Known in the prior art. The strong but balanced cellular and humoral immune responses resulting from CpG stimulation reflect the body's own natural defense system against invading pathogenic microorganisms and cancer cells. CpG sequences that are relatively rare in human DNA are commonly found in DNA of infectious microorganisms such as bacteria. The human immune system is clearly C
It evolved to recognize the pG sequence as an early warning sign of infection and to generate a rapid and powerful immune response against invading pathogenic microorganisms without the side effects often seen with other immunostimulants. Therefore, CpG-containing nucleic acids that depend on this inactive immune defense mechanism are:
A unique and natural route can be used for immunotherapy.

The effect of CpG nucleic acids on immune modification was discovered by the inventors of the present patent application and has been extensively described in co-pending patent applications. Such co-pending applications include US patent application Ser. No. 08 / 386,063 (filing date 02/07/95) (and related PCT).
US95 / 01570), 08 / 738,652 (filing date 10/30/96), 0
8 / 960,774 (filing date 10/30/97) (and related PCT applications / US 97 /
19791, WO 98/18810), 09 / 191,170 (filing date 11/13)
/ 98), 09 / 030,701 (filing date 02/25/98) (and related PC / U
S98 / 03678, 09 / 082,649 (filing date 05/20/98) (and related PC / US98 / 10408), 09 / 325,193 (filing date 06/03/99)
(And related PC / US98 / 04703), 09 / 286,098 (filing date 04
/ 02/99) (and related PC / US99 / 07335), 09 / 306,281 (filing date 05/06/99) (and related PCT / US99 / 09863). The entire contents of each of these patents and patent applications are hereby incorporated by reference.

The present invention is based, in part, on the unexpected discovery of several families of nucleic acids that are more immunostimulatory than previously reported CpG nucleic acids. Each family is represented by a particularly immunostimulatory nucleic acid. These nucleic acid families and their representative members are
More details are described below.
ODN10102 Family The nucleic acid _{family, 5'X 1 X 2 X 3 X} 4 X 5 X 6 X 7 TT CGT CGT
Consisting of a nucleotide sequence having the formula TTC GTC GTT 3 ′ (SEQ ID NO: 3),
Here, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , and X ₇ are each independently selected residues, which are adenosine, guanosine, thymidine, and cytosine. Can be selected from the nucleotide group consisting of In some embodiments, flanking residues may not be present. Such a nucleic acid is 5′TT CGT CGT TTC GTC
Contains the nucleotide sequence of GTT3 ′ (SEQ ID NO: 4).

In other embodiments, the nucleic acid comprises: X ₁ ; X ₁ and X ₂ ; X ₁ , X ₂ , and X ₃ ; X ₁ , X ₂ , X
₃ and X ₄ ; or X ₁ , X ₂ , X ₃ , X ₄ and X ₅ may be absent, or X ₁ to X ₆
Or it may lack up to, or from X ₁ to X ₇ may lack.

In one embodiment, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X
₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine, and / or X ₆ is guanosine, and / or X ₁ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This family of nucleic acids is generally at least 17 nucleotides in length. In some embodiments, the nucleic acid is at least 19, at least 20, at least 21, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is 24
Exceeds nucleotide length. Examples are at least 50, at least 75, at least 1
Nucleic acids that are 00, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 17-100, more preferably 24-100 nucleotides in length.

All of this first family of nucleic acids contain at least three CpG motifs. These nucleic acids may contain 4 or 5 or more CpG motifs. CpG motifs are adjacent to each other, or alternately, at a certain distance or any distance from each other,
They may be separated from each other.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
It may contain thymidine above 60%, below 60%, or below 55%.

The present invention is further premised on, in part, the unexpected discovery of another nucleic acid family that is more immunostimulatory than previously reported CpG nucleic acids. This nucleic acid family has the formula 5 ′
_{TCG TCG TTT CGT CGT TTC X 1} X 2 X 3 X 4 X 5 X 6 3 ' is composed of the nucleotide sequence having the formula (SEQ ID NO: 5), In the _{formula, X 1, X 2, X} 3, X
₄ , X ₅ , and X ₆ are independently selected residues that can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. Such nucleic acids are 5
It contains the nucleotide sequence of 'TCG TCG TTT CGT CGT TTC3' (SEQ ID NO: 6).

In other embodiments, the nucleic acid is lack of X ₆ , lack of X ₆ and X ₅ , or X ₆ , X ₅ ,
And lack of _{X 4,} the lack of the _{X 6} to _{X 3,} lack from _{X 6} to _{X 2,} or may be those lacking from _{X 6} to _{X 1} has occurred. .

In one embodiment, X ₁ is cytosine. In another embodiment, X ₂ is guanosine. In another embodiment, X ₃ is thymidine. In another embodiment, X ₄ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This latter family of nucleic acids is generally at least 18 nucleotides in length. In some embodiments, the nucleic acid is at least 20, at least 22, at least 23,
And at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids that are at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 18-100, more preferably 24-100 nucleotides in length.

All of this second family of nucleic acids contain at least four CpG motifs. These nucleic acids may contain 5 or more CpG motifs depending on the embodiment. CpG motifs may be adjacent to each other or alternately spaced apart from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
It may contain thymidine above 60%, below 60%, or below 55%.

In another aspect, the present invention provides TCG TCG TTT CGT CGT TTC GTC
Nucleic acids comprising the nucleotide sequence of GTT (SEQ ID NO: 1) (ODN 10102) are provided. As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for an immunostimulatory activity similar to or better than the previously identified immunostimulatory nucleic acids. More specifically, this nucleic acid was compared to a nucleic acid having a nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) that has already been shown to be immunostimulatory. Nucleic acids containing SEQ ID NO: 1 were identified only after screening about 165 nucleic acids for those with greater immunostimulatory capacity than those containing SEQ ID NO: 2. The difference in activity is surprising. This is because there is only a minimal difference (ie, a difference between two nucleotides) between SEQ ID NO: 1 and SEQ ID NO: 2. It was unexpected that minimal changes in such sequences resulted in a statistically significant increase in immune stimulation.

In still another aspect of the present invention, a nucleic acid having the following nucleotide sequence is provided. That is, 5′TCG TCG TTT CGT CGT TTC GTC GT3 ′ (SEQ ID NO: 7), 5′TCG TCG TTT CGT CGT TTC GTC G3 ′ (
SEQ ID NO: 8), 5'TCG TCG TTT CGT CGT TTC GTC3 '(SEQ ID NO: 9), 5'TCG TCG TTT CGT CGT TTC GT3' (SEQ ID NO: 10), 5'TCG TCG TTT CGT CGT TTC ' 1
1) 5′CG TCG TTT CGT CGT TTC GTC GTT3 ′ (SEQ ID NO: 12), 5′G TCG TTT CGT CGT TTC GTC GTT3 ′ (
SEQ ID NO: 13), 5′TCG TTT CGT CGT TTC GTC GTT3 ′ (
SEQ ID NO: 14), 5′CG TTT CGT CGT TTC GTC GTT3 ′ (SEQ ID NO: 15), 5′G TTT CGT CGT TTC GTC GTT3 ′ (SEQ ID NO: 16), 5′TTT CGT CGT TTC GTC GTT3 ′ (SEQ ID NO: 17) )
, And 5'TT CGT CGT TTC GTC GTT 3 '(SEQ ID NO: 18).

The nucleic acid of the present invention contains other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequences of SEQ ID NO: 4 and SEQ ID NO: 6 exist. Further can be included. These immunostimulatory motifs are described below or in US application 09 / 669,187 (filed September 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972), It is described in more detail.

(ODN10103 family)
This nucleic acid family is 5′X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X _1
1 X ₁₂ GGT CGT TTT 3 ′ (SEQ ID NO: 20) consisting of a nucleotide sequence, wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ , X ₉ , X _1
0 , X ₁₁ , and X ₁₂ are independently selected residues, which can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. Such a nucleic acid comprises the nucleotide sequence of 5 ′ GGT CGT TTT 3 ′ (SEQ ID NO: 21).

In other embodiments, the nucleic acid, the absence of _{X 1,} the lack of _{X 1} and _{X 2,} the lack of X 1, the lack of _{X 2,} and _{X 3,} X _1, X 2, _{X 3,} and _{X 4,} or , X ₁ , X ₂ , X ₃ , X ₄ ,
And X ₅ missing, X ₁ to X ₆ missing, X ₁ to X ₇ missing, X ₁ to X ₈ missing, X ₁ to X ₉ missing, X ₁ to X ₁₀ missing , X ₁ to X ₁₁ , and X ₁ to X ₁₂ .

In various embodiments, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X ₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine,
And / or X ₆ is guanosine, and / or X ₇ is thymidine, and / or X ₈
Is thymidine, and / or X ₉ is thymidine, and / or X ₁₀ is thymidine, and / or X ₁₁ is thymidine, and / or X ₁₂ is cytosine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This family of nucleic acids is generally at least 9 nucleotides in length. In some embodiments, the nucleic acid is at least 10, at least 12, at least 15, at least 18, at least 20, and at least 21 nucleotides in length. In a preferred embodiment, the nucleic acid is 21 nucleotides in length. In yet another embodiment, the nucleic acid is greater than 21 nucleotides in length. As an example, at least 50, at least 75, at least 100,
Nucleic acids that are at least 200, at least 500, at least 1,000 nucleotides in length or longer are included. Preferably, the nucleic acid is 9-100, more preferably 21-1
It is 00 nucleotides long.

All of the nucleic acids of this first family contain at least one CpG motif. These nucleic acids may contain 2, 3, 4, or more CpG motifs. The CpG motifs may be adjacent to each other, or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
It may comprise at least 60%, at least 55%, or at least 50% thymidine.

The present invention is further premised on, in part, the unexpected discovery of another nucleic acid family that is immunostimulatory equivalent to previously reported CpG nucleic acids. This nucleic acid family is 5 '
It consists _{TCG TCG TTT TTC X 1 X 2} X 3 X 4 X 5 X 6 X 7 X 8 X 9 3 ' nucleotide sequence having the formula (SEQ ID NO: 22), _{X 9} in formula, _{X 1} to the Each is an independently selected residue, which can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. By way of example, this nucleic acid is 5′TCG T
It may comprise the nucleotide sequence of CG TTT TTC 3 ′ (SEQ ID NO: 23).

In other embodiments, the nucleic acid, the absence of _{X 9,} lack of _{X 9} and _{X 8,} lack of X 9, _{X 8,} and _{X 7,} lack from _{X 9} to _{X 6,} from _{X 9} in _{X 5} lack, lack from _{X 9} to _{X 4,} the lack of the _{X 9} to _{X 3,} it may be one with a lack of lack from _{X 9} to _{X 2,} and from _{X 9} to _{X 1.}

In various embodiments, X ₁ is guanosine, and / or X ₂ is guanosine, and / or
Or X ₃ is thymidine, and / or X ₄ is cytosine, and / or X ₅ is guanosine, and / or X ₆ is thymidine, and / or X ₇ is thymidine, and / or X
₈ thymidine, and / or _{X 9} is a thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This family of nucleic acids is generally at least 12 nucleotides in length. In some embodiments, the nucleic acid is at least 15, at least 18, at least 21 nucleotides in length. In a preferred embodiment, the nucleic acid is 21 nucleotides in length. In still further embodiments, the nucleic acid is greater than 21 nucleotides in length. By way of example, at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,
Nucleic acids that are 000 nucleotides in length or longer. Preferably, the nucleic acid is 1
The length is 2 to 100, more preferably 21 to 100 nucleotides.

All of this second family of nucleic acids contain at least two CpG motifs. These nucleic acids may contain 3 or 4 or more CpG motifs,
Depends on the embodiment. The CpG motifs may be adjacent to each other, or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
It may comprise at least 60%, at least 55%, or at least 50% thymidine.

In another aspect, the present invention provides TCG TCG TTT TTC GGT CGT TTT
A nucleic acid comprising the nucleotide sequence of (SEQ ID NO: 19) (ODN 10103) is provided. As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for nucleic acid having an immunostimulatory activity similar to or better than previously identified immunostimulatory nucleic acids. . More specifically, this nucleic acid was compared to a nucleic acid having the nucleotide sequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2) previously shown to be immunostimulatory. A nucleic acid comprising SEQ ID NO: 19 was identified only after screening about 165 nucleic acids for immunostimulatory ability similar to or greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is a minimal difference between SEQ ID NO: 19 and SEQ ID NO: 2 (ie, SEQ ID NO: 19 compared to SEQ ID NO: 2 has three additional internal nucleotides (ie, TCG)
And 6 3 ′ nucleotides are deleted). It was unexpected that such changes in the sequence resulted in increased immune stimulation.

In yet another aspect of the invention, nucleic acids having the following nucleotide sequences are provided. That is, 5′TCG TCG TTT TTC GGT CGT TT3 ′ (SEQ ID NO: 24
) 5′TCG TCG TTT TTC GGT CGT T3 ′ (SEQ ID NO: 25),
5′TCG TCG TTT TTC GGT CGT3 ′ (SEQ ID NO: 26), 5′TC
G TCG TTT TTC GGT CG 3 ′ (SEQ ID NO: 27), 5 ′ TCG TCG
TTT TTC GGT C3 ′ (SEQ ID NO: 28), 5′TCG TCG TTT T
TC GGT3 ′ (SEQ ID NO: 29), 5′TCG TCG TTT TTC GG3 ′ (
SEQ ID NO: 30), 5 ′ TCG TCG TTT TTC G3 ′ (SEQ ID NO: 44), 5 ′
TCG TCG TTT TTC 3 ′ (SEQ ID NO: 31), 5 ′ TCG TCG TTT
TTC GGT CGT TTT 3 ′ (SEQ ID NO: 32), 5 ′ CG TCG TTT T
TC GGT CGT TTT 3 ′ (SEQ ID NO: 33), 5 ′ G TCG TTT TTC
GGT CGT TTT 3 ′ (SEQ ID NO: 34), 5 ′ TCG TTT TTC GGT
CGT TTT 3 ′ (SEQ ID NO: 35), 5 ′ CG TTT TTC GGT CGT
TTT3 ′ (SEQ ID NO: 36) 5′G TTT TTC GGT CGT TTT3 ′ (SEQ ID NO: 37), 5′TTT TTC GGT CGT TTT3 ′ (SEQ ID NO: 38), 5
'TT TTC GGT CGT TTT3' (SEQ ID NO: 39), 5'T TTC GG
T CGT TTT 3 ′ (SEQ ID NO: 40), 5 ′ TTC GGT CGT TTT 3 ′
(SEQ ID NO: 41) 5′TC GGT CGT TTT 3 ′ (SEQ ID NO: 42), 5′C
GGT CGT TTT 3 ′ (SEQ ID NO: 43), 5 ′ GGT CGT TTT 3 ′ (SEQ ID NO: 21).

These immunostimulatory nucleic acids have the ability to activate the innate immune system and enhance both humoral and cellular antigen-specific responses when antigens such as hepatitis B surface antigen are co-administered Have the ability to The examples provided herein show that these nucleic acids are in vitro.
o) shows that human immune cells can be stimulated, and mouse cells can be stimulated in vitro and in vivo. When compared to a sequence known to be a potent adjuvant, the nucleic acid of SEQ ID NO: 19 is at least 10-15% as a vaccine adjuvant.

The nucleic acid of the present invention contains other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequences of SEQ ID NO: 21 and SEQ ID NO: 23 exist. Further can be included. These immunostimulatory motifs
The description below or US application 09 / 669,187 (filed September 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO01 / 22972).
).

(ODN 10104 family)
This nucleic acid family is 5′X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ TTT CGT CGT T
Consisting of a nucleotide sequence having the formula TT GTC GTT 3 ′ (SEQ ID NO: 46), wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , and X ₆ are independently selected residues And the residue can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. Such a nucleic acid is 5 ′ TTT CGT CGT TTT GTC GTT3
The nucleotide sequence of '(SEQ ID NO: 47) is included.

In other embodiments, the nucleic acid, the absence of _{X 1,} the lack of _{X 1} and _{X 2,} the lack of X 1, the lack of _{X 2,} and _{X 3,} X _1, X 2, _{X 3,} and _{X 4,} or It may be accompanied by a lack of X ₁ , X ₂ , X ₃ , X ₄ , and X ₅ . Accordingly, the present invention is intended to encompass nucleic acids having the following nucleotide sequences: That is, 5′X ₂ X ₃ X ₄ X ₅ X
₆ TTT CGT CGT TTT GTC GTT 3 ′ (SEQ ID NO: 48), 5 ′ X ₃
X ₄ X ₅ X ₆ TTT CGT CGT TTT GTC GTT 3 ′ (SEQ ID NO: 49)
_{, 5'X 4 X 5 X 6 TTT} CGT CGT TTT GTC GTT3 '( SEQ ID NO: _{50), 5'X 5 X 6 TTT} CGT CGT TTT GTC GTT3' ( SEQ ID NO: _{51), 5'X 6 TTT CGT CGT} TTT GTC GTT3 ′ (SEQ ID NO: 52).

In one embodiment, X ₁ is thymidine. In another embodiment, X ₂ is cytosine.
In another embodiment, X ₃ is guanosine. In another embodiment, X ₄ is thymidine. In yet another embodiment, X ₅ is cytosine. In yet another embodiment, X ₆ is guanosine. The invention further encompasses combinations of multiple flanking residues as follows (blank cells are N residues, ie, naturally occurring as described herein or known in the art) Or any non-naturally occurring nucleotide).

Table 1 represents only some of the possible nucleic acids that are members of the first nucleic acid family. One skilled in the art can determine the sequence of the remaining nucleic acids belonging to this family.

This family of nucleic acids is generally at least 18 nucleotides in length. In some embodiments, the nucleic acid is at least 19, at least 20, at least 21, at least 22, at least 23, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is 2
Over 4 nucleotides in length. Examples include at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length,
Or a longer nucleic acid is mentioned. Preferably, the nucleic acid is 18-100, more preferably 24-100 nucleotides in length.

All of this first family of nucleic acids contain at least three CpG motifs. These nucleic acids may contain 4 or 5, or more CpG motifs. The CpG motifs may be adjacent to each other, or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
It may contain more than 60%, less than 60%, or less than 55% thymidine.

The present invention is further premised on the unexpected discovery of another nucleic acid family that is more immunostimulatory than previously reported CpG nucleic acids. This nucleic acid family is 5'T
Consists CG TCG TTT CGT CGT TTT GT X 1 X 2 X 3 X 4 3 ' nucleotide sequence having the formula (SEQ ID NO: 95), In the _{formula, X 1, X 2, X} 3, and _{X 4,} An independently selected residue, which can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. By way of example, the nucleic acid is 5′TCG TC
G TTT CGT, CGT, including the nucleotide sequence of TTT GT 3 ′ (SEQ ID NO: 96).

In other embodiments, the nucleic acid, the absence of _{X 4,} the lack of _{X 4} and _{X 3,} the lack of _{X 4} and _{X 3,} or _X 4, _{X 3,} and lack of _{X 2} is even arose Good. Accordingly, the present invention is intended to encompass nucleic acids having the following nucleotide sequences: That is,
5′TCG TCG TTT CGT CGT TTT GT X ₁ X ₂ X ₃ 3 ′ (SEQ ID NO: 97) 5 ′ TCG TCG TTT CGT CGT TTT GT X ₁ X ₂ 3
'(SEQ ID NO: 98), and 5' TCG TCG TTT CGT CGT TTT G
T X ₁ 3 ′ (SEQ ID NO: 99).

In one embodiment, X ₁ is cytosine. In another embodiment, X ₂ is guanosine. In another embodiment, X ₃ is thymidine. In another embodiment, X ₄ is thymidine. The invention further encompasses combinations of multiple flanking residues as follows (blank cells are N residues, ie, naturally occurring or naturally occurring, as described or known herein). Any of the nucleotides not present in).

Table 2 represents only some of the possible nucleic acids that are members of the second nucleic acid family. One skilled in the art can determine the sequence of the remaining nucleic acids belonging to this family.

This latter family of nucleic acids is generally at least 20 nucleotides in length. In some embodiments, the nucleic acid is at least 21, at least 22, at least 23,
And at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples include nucleic acids that are at least 50, at least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 20-100, more preferably 24-100 nucleotides in length.

All of this second family of nucleic acids contain at least four CpG motifs. These nucleic acids may contain 5 or more CpG motifs, depending on the embodiment. C
The pG motifs may be adjacent to each other, or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids are
More than 60% or less than 60% or less than 55% thymidine can be included.

In another aspect, the present invention provides TCG TCG TTT CGT CGT TTT GTC
Nucleic acids comprising the nucleotide sequence of GTT (SEQ ID NO: 45) (ODN 10104) are provided. As described in more detail in the examples, this nucleic acid was identified only after screening a large number of nucleic acids for an immunostimulatory activity similar to or better than previously identified immunostimulatory nucleic acids. More specifically, the nucleic acid is converted to TCG TCG TTT TGT CGT TTT GTC GT previously shown to be immunostimulatory.
Comparison was made with a nucleic acid having the nucleotide sequence of T (SEQ ID NO: 2). A nucleic acid comprising SEQ ID NO: 45, a nucleic acid having an immunostimulatory capacity greater than that of the nucleic acid comprising SEQ ID NO: 2,
Only about 165 nucleic acids were identified after screening. The difference in activity is surprising. This is because there is only a minimal difference between SEQ ID NO: 45 and SEQ ID NO: 2 (ie, replacement of thymidine (SEQ ID NO: 2) by cytosine (SEQ ID NO: 45)). This minimal change in sequence results in a statistically significant increase in immunostimulatory properties,
It was unexpected.

In yet another aspect of the invention, nucleic acids having the following nucleotide sequences are provided. That is, 5′CG TCG TTT CGT CGT TTT GTC GTT3 ′ (SEQ ID NO: 110), 5′G TCG TTT CGT CGT TTT GTC GTT3 ′
(SEQ ID NO: 111) 5'TCG TTT CGT CGT TTT GTC GTT3
'(SEQ ID NO: 112), 5' CG TTT CGT CGT TTT GTC GTT3
'(SEQ ID NO: 113), 5'G TTT CGT CGT TTT GTC GTT3'
(SEQ ID NO: 114), 5 ′ TTT CGT CGT TTT GTC GTT 3 ′ (SEQ ID NO: 47), 5 ′ TCG TCG TTT CGT CGT TTT GTC GT 3
'(SEQ ID NO: 115), 5'TCG TCG TTT CGT CGT TTT GTC
G3 ′ (SEQ ID NO: 116), 5′TCG TCG TTT CGT CGT TTT
GTC 3 ′ (SEQ ID NO: 117), 5 ′ TCG TCG TTT CGT CGT TTT
GT3 ′ (SEQ ID NO: 96).

The nucleic acid of the present invention contains other immunostimulatory motifs such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequences of SEQ ID NO: 47 and SEQ ID NO: 96 exist. Further can be included. These immunostimulatory motifs
The description below or US application 09 / 669,187 (filed September 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO01 / 22972).
) In more detail.

(ODN 10105 family)
This nucleic acid family is 5′X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X _1
1 X ₁₂ X ₁₃ X ₁₄ X ₁₅ TTT TTT CGA 3 ′ (SEQ ID NO: 119) consisting of a nucleotide sequence, wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ ,
X ₇ , X ₈ , X ₉ , X ₁₀ , X ₁₁ , X ₁₂ , X ₁₃ , X ₁₄ , and X ₁₅ are independently selected residues, which are adenosine, guanosine, thymidine, And a nucleotide group consisting of cytosine. In some embodiments, flanking residues may not be present. Such a nucleic acid is 5 ′ TTT TTT CGA 3 ′ (
SEQ ID NO: 120).

In other embodiments, the nucleic acid, the absence of _{X 1,} the lack of _{X 1} and _{X 2,} the lack of X 1, the lack of _{X 2,} and _{X 3,} X _1, X 2, _{X 3,} and _{X 4,} or X ₁ , X ₂ , X ₃ , X ₄ ,
And lack of _{X 5,} lack of lack from _{X 1} to _{X 6,} the lack of the _{X 1} to _{X 7,} the lack of the _{X 1} to _{X 8,} the lack of the _{X 1} to _{X 9, X 1 to} to _{X 10} , X ₁ to X ₁₁ , X ₁ to X ₁₂ , X ₁ to X ₁₃ , X ₁ to X ₁₄ , and X ₁ to X _15. There may be.

In various embodiments, X ₁ is thymidine, and / or X ₂ is cytosine, and / or X ₃ is guanosine, and / or X ₄ is thymidine, and / or X ₅ is cytosine,
And / or X ₆ is guanosine, and / or X ₇ is thymidine, and / or X ₈
Is thymidine, and / or X ₉ is thymidine, and / or X ₁₀ is thymidine, and / or X ₁₁ is guanosine, and / or X ₁₂ is thymidine, and / or X ₁₃
Is cytosine, and / or X ₁₄ is guanosine, and / or X ₁₅ is thymidine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This family of nucleic acids is generally at least 9 nucleotides in length. In some embodiments, the nucleic acid is at least 10, at least 12, at least 15, at least 18, at least 20, at least 22, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. Examples are at least 50, at least 75,
Examples include nucleic acids that are at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 9-100, more preferably 24-100 nucleotides in length.

All of the nucleic acids of this first family contain at least one CpG motif. These nucleic acids may contain 2, 3, 4 or more CpG motifs. C
The pG motifs may be adjacent to each other or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

The present invention is further premised on, in part, the unexpected discovery of another nucleic acid family that is immunostimulatory similar to previously reported CpG nucleic acids. This nucleic acid family is 5 '
TCG TCG TTT TGT CGT TTT TX ₁ X ₂ X ₃ X ₄ X ₅ 3 '
It is composed of a nucleotide sequence having the formula (SEQ ID NO: 121), wherein X ₁ to X ₉
Are each independently selected residues, which can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine. In some embodiments, flanking residues may not be present. By way of example, such a nucleic acid is 5 ′
It includes the nucleotide sequence of TCG TCG TTT TGT CGT TTT T 3 ′ (SEQ ID NO: 122).

In other embodiments, the nucleic acid, the absence of _{X 5,} lack of _{X 5} and _{X 4,} the lack of X 5, _{X 4,} and _{X 3,} the lack of the _{X 5} to _{X 2,} and from _{X 5} to _{X 1} The lack of may have occurred.

In various embodiments, X ₁ is thymidine and / or X ₂ is thymidine;
And / or X ₃ is cytosine and / or X ₄ is guanosine and / or X ₅ is adenine. One skilled in the art can determine the remaining nucleic acid sequences belonging to this family.

This family of nucleic acids is generally at least 19 nucleotides in length. In some embodiments, the nucleic acid is at least 20, at least 22, and at least 24 nucleotides in length. In a preferred embodiment, the nucleic acid is 24 nucleotides long. In still further embodiments, the nucleic acid is greater than 24 nucleotides in length. As an example, at least 50
, At least 75, at least 100, at least 200, at least 500, at least 1,000 nucleotides in length, or longer. Preferably, the nucleic acid is 19-100, more preferably 24-100 nucleotides in length.

All of this second family of nucleic acids contain at least three CpG motifs. These nucleic acids may contain four or more CpG motifs depending on the embodiment.
CpG motifs may be adjacent to each other or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids can comprise at least 60%, at least 55%, or at least 50% thymidine.

In another aspect, the present invention provides TCG TCG TTT TGT CGT TTT TTT
Nucleic acids composed of nucleotide sequences having the formula CGA (SEQ ID NO: 118) (ODN 10105) are provided. As described in more detail in the Examples, this nucleic acid was identified only after screening a large number of nucleic acids for an immunostimulatory activity similar to or better than the previously identified immunostimulatory nucleic acids. More specifically, this nucleic acid is converted to TCG TCG TTT TGT CGT TTT G that has already been shown to be immunostimulatory.
Comparison was made with a nucleic acid having a nucleotide sequence of TC GTT (SEQ ID NO: 2). SEQ ID NO: 118
Was identified only after screening about 165 nucleic acids for immunostimulatory activity similar to or greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is 79% identity between SEQ ID NO: 118 and SEQ ID NO: 2 (that is, 5 nucleotides on the 3 ′ side are different between SEQ ID NO: 118 and SEQ ID NO: 2). It was unexpected that changes in the sequence could lead to increased immune stimulation.

In still another embodiment of the present invention, a nucleic acid having the following nucleotide sequence is obtained. That is,
5 ′ TCG TCG TTT TGT CGT TTT TTT CG 3 ′ (SEQ ID NO: 123) 5 ′ TCG TCG TTT TGT CGT TTT TTT C 3
'(SEQ ID NO: 124), 5' TCG TCG TTT TGT CGT TTT TT
T 3 ′ (SEQ ID NO: 125), 5 ′ TCG TCG TTT TGT CGT TTT
TT 3 ′ (SEQ ID NO: 126), 5 ′ CG TCG TTT TGT CGT TT
T TTT CGA 3 ′ (SEQ ID NO: 127), 5 ′ G TCG TTT TGT C
GT TTT TTT CGA 3 ′ (SEQ ID NO: 128), 5 ′ TCG TTT TG
T CGT TTT TTT CGA 3 ′ (SEQ ID NO: 129), 5 ′ CG TTT
TGT CGT TTT TTT CGA 3 ′ (SEQ ID NO: 130), 5 ′ G TTT
TGT CGT TTT TTT CGA 3 ′ (SEQ ID NO: 131), 5 ′ TTT
TGT CGT TTT TTT CGA 3 ′ (SEQ ID NO: 132), 5 ′ TT TG
T CGT TTT TTT CGA 3 ′ (SEQ ID NO: 133), 5 ′ T TGT C
GT TTT TTT CGA 3 '(SEQ ID NO: 134), 5' TGT CGT TT
T TTT CGA 3 ′ (SEQ ID NO: 135), 5 ′ GT CGT TTT TTT
CGA 3 ′ (SEQ ID NO: 136), 5 ′ T CGT TTT TTT CGA 3 ′ (
SEQ ID NO: 137), 5 ′ CGT TTT TTT CGA 3 ′ (SEQ ID NO: 138),
5 ′ GT TTT TTT CGA 3 ′ (SEQ ID NO: 139), and 5 ′ T TT
T TTT CGA 3 '(SEQ ID NO: 140).

These immunostimulatory nucleic acids have the ability to activate the innate immune system and enhance both humoral and cellular antigen-specific responses when antigens such as hepatitis B surface antigen are co-administered . The examples provided herein show that these nucleic acids can stimulate human immune cells in vitro and in vitro and in vivo.
Demonstrate that iv) can stimulate mouse cells. When compared to a sequence known to be a potent adjuvant, it can be seen that the nucleic acid of SEQ ID NO: 118 acts equivalently or better as a vaccine adjuvant.

The nucleic acid of the present invention has another immunostimulatory motif such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequences of SEQ ID NO: 120 and SEQ ID NO: 122 are present. Further can be included. These immunostimulatory motifs are described below or in US application 09 / 669,187 (filed 25 September 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO01 / 229).
72).

(ODN 10106 family)
The nucleic acid comprises a nucleic acid sequence having the formula TCG TCG TTT TTC GTG CGT TTT T (SEQ ID NO: 141) (ODN 10106).

The sequence can be flanked by a number of independently selected nucleotide residues that can be selected from the group of nucleotides consisting of adenosine, guanosine, thymidine, and cytosine.

This family of nucleic acids is at least 22 nucleic acids in length. In a preferred embodiment, the nucleic acid is 22 nucleotides long. In still further embodiments, the nucleic acid is greater than 22 nucleotides in length. For example, at least 50, at least 75, at least 100, at least 2
00, at least 500, at least 1,000 nucleotides in length, or longer nucleic acids. Preferably, the nucleic acid is 12-100.

All of the nucleic acids of this first family contain at least 4 CpG motifs. These nucleic acids may contain 5 or more CpG motifs. The CpG motif is
They may be adjacent to each other or they may be separated from each other by a fixed distance or any distance.

This family of nucleic acids also includes overexpression of thymidine nucleotides. These nucleic acids can contain greater than 60%, less than 60%, or less than 55% thymidine.

In another aspect, the present invention provides TCG TCG TTT TTC GTG CGT TTT
A nucleic acid comprising the nucleotide sequence of T (SEQ ID NO: 141) is provided. As explained in more detail in the examples, this nucleic acid was identified only after screening a large number of nucleic acids for an immunostimulatory activity similar to or better than the previously identified immunostimulatory nucleic acids. More specifically, this nucleic acid is a TCG T that has already been shown to be immunostimulatory.
Comparison was made with a nucleic acid having the nucleotide sequence of CG TTT TGT CGT TTT GTC GTT (SEQ ID NO: 2). A nucleic acid comprising SEQ ID NO: 141 was identified only after screening about 165 nucleic acids for an immunostimulatory capacity greater than that of the nucleic acid comprising SEQ ID NO: 2. The difference in activity is surprising. This is because there is only a minimum difference between the sequence code 141 and the sequence number 2. It was unexpected that such minimal changes in sequence resulted in a statistically significant increase in immune stimulation.

The nucleic acid of the present invention may further contain another immunostimulatory motif such as poly T motif, poly G motif, TG motif, poly A motif, poly C motif, etc., provided that the core sequence of SEQ ID NO: 141 exists. it can. These immunostimulatory motifs are described in more detail in the description below or in US application 09 / 669,187 (filed 25 September 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO 01/22972). It is described in.

It is understood that any embodiment detailed herein is equivalent to the embodiments and applies to the nucleic acids provided herein. Thus, for example, if an embodiment refers to SEQ ID NO: 1, it is understood that it applies equally to SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118, and SEQ ID NO: 141.

The CpG motif of the nucleic acids described herein is preferably not methylated. An unmethylated CpG motif is an unmethylated cytosine-guanine dinucleotide sequence (ie, an unmethylated 5 ′ cytosine followed by a 3 ′ guanosine and joined by a phosphate bond). All of the nucleic acids described herein are immunostimulatory. In some embodiments of the invention, the CpG motif is methylated. A methylated CpG motif is a methylated cytosine-guanine dinucleotide sequence (ie, a methylated 5 ′ cytosine followed by a 3 ′ guanosine and linked by a phosphate bond).

CpG nucleic acids have the formula:
5 ′ X ₁ X ₂ CGX ₃ X ₄ 3 ′
Wherein C is not methylated and X ₁ X ₂ and X ₃ X ₄ are nucleotides. In a related embodiment, the 5 ′ X ₁ X ₂ CGX ₃ X ₄ 3 ′ sequence is a non-palindromic sequence. In certain embodiments, X ₁ X ₂ is GpT, GpG, GpA, A
a nucleotide selected from the group consisting of pA, ApT, ApG, CpT, CpA, CpG, TpA, TpT, and TpG, and X ₃ X ₄ is TpT, CpT, ApT,
A nucleotide selected from the group consisting of TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, and CpA. In a more specific embodiment, X ₁ X ₂ is a nucleotide selected from the group consisting of GpA and GpT, and X ₃ X ₄
Is TpT. In yet another embodiment, X ₁ X ₂ are both purines and X ₃ X
Both ₄ are pyrimidines. In another embodiment, X ₂ is T and X ₃ is a pyrimidine. Examples of CpG nucleic acids are described in US Application No. 09 / 669,187 (filed September 25, 2000) and published PCT patent application PCT / US00 / 26383 (publication number WO01 / 22).
972).

A T-rich nucleic acid is a nucleic acid comprising at least one poly T sequence and / or having a nucleotide composition with more than 25% T nucleotide residues. A nucleic acid having a poly-T sequence contains at least 4 Ts in succession, for example, 5′TTTT3 ′. Preferably, the T-rich nucleic acid includes one or more poly T sequences. In preferred embodiments, the T-rich nucleic acid may have 2, 3, 4, or some other number of poly T sequences. Other T-rich nucleic acids according to the present invention have a nucleotide composition of more than 25% T nucleotide residues, but it is not essential to include a poly T sequence. In these T-rich nucleic acids, other types of nucleotide residues (ie, G, C, and A) separate T nucleotide residues from each other. In some embodiments, T-rich nucleic acids contain 35%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% of T nucleotide residues, and any integer% in between. Has a higher nucleotide composition. Preferably, the T-rich nucleic acid has at least one poly T sequence and a nucleotide composition with more than 25% T nucleotide residues.

The poly G nucleic acid is preferably a nucleic acid having the following formula: That is,
5 ′ X ₁ X ₂ GGGX ₃ X ₄ 3 ′
Where X ₁ , X ₂ , X ₃ , and X ₄ are nucleotides. In a preferred embodiment,
At least one of X ₃ and X ₄ is G. In other embodiments, both X ₃ and X ₄ are G. In yet another embodiment, the preferred formula is 5 ′ GGGNGGG 3 ′.
Or 5 ′ GGGNGGGNGGG 3 ′, where N represents between 0 and 20 nucleotides.

A C-rich nucleic acid is a nucleic acid molecule that has at least one or preferably at least two poly C regions, or is composed of at least 50% C nucleotides. Poly C
A region is at least 4 consecutive C residues. Thus, the poly C region is expressed by the equation 5′C
It is included in CCC3 ′. In some embodiments, the poly C region is of formula 5′CCCCCC3 ′.
It is preferable to have. Other C-rich nucleic acids according to the present invention have a nucleotide composition with more than 50% C nucleotide residues, but it is not essential to include a poly C sequence. In these C-rich nucleic acids, other types of nucleotide residues (ie, G, T, and A)
Can separate C nucleotide residues from each other. In some embodiments, the C-rich nucleic acid has a nucleotide composition with more than 60%, 70%, 80%, 90%, and 99% C nucleotide residues, and any integer percentage in between. Preferably, the C-rich nucleic acid has at least one poly C sequence and a nucleotide composition with more than 50% C nucleotide residues, and in some embodiments is also T-rich.

Immunostimulatory nucleic acids are double stranded or single stranded. Usually, double-stranded molecules are in vivo (in v
While more stable in ivo), single-stranded molecules have high immune activity. Thus, in some aspects of the invention, it is preferred that the nucleic acid is single stranded, and in other aspects, the nucleic acid is double stranded.

The terms “nucleic acid” and “oligonucleotide” are used interchangeably herein, and bind to a phosphate group and are substituted pyrimidines (eg, cytosine (C), thymidine (
T), or uracil (U)) or substituted purines (eg, adenine (A) or guanine (G)). Molecule) containing deoxyribose). As used herein, the term refers to oligoribonucleotides as well as oligodeoxyribonucleotides. The term also includes polynucleosides (eg, polynucleotides from which phosphate has been removed) and other organic base-containing polymers. Nucleic acid molecules can be obtained from existing nucleic acid sources (eg, genomic DNA or cDNA), but are preferably synthesized (eg, produced by nucleic acid synthesis).

The immunostimulatory oligonucleotides of the present invention include various chemical modifications and substitutions compared to natural RNA and DNA, and include internucleoside cross-linking with phosphodiester, β-D-ribose units, and / or Or natural nucleoside bases (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to those skilled in the art,
For example, Uhlmann E et al. (1990) Chem Rev 90: 543; “Prot
ocols for Oligonucleotides and Analogs "S
synthesises and properties & synthesises and
Analytical Technologies, S.A. Agrawal (edit) Human
a Press, Totowa, USA 1993; Crooke ST et al. (1996).
Annu Rev Pharmacol Toxicol 36: 107-129; and Hunziker J et al. (1995) Mod Synth Methods 7:33.
1-417. Oligonucleotides according to the present invention may have one or more modifications, each modification being a specific phosphodiester compared to an oligonucleotide of the same sequence composed of natural DNA or RNA. • Located at internucleoside bridges and / or β-D-ribose units and / or at certain natural nucleoside base positions.

For example, the oligonucleotide may include one or more modifications, each modification being independently
a) substitution of the phosphodiester internucleoside bridge located at the 3 ′ and / or 5 ′ end of the nucleoside by a modified internucleoside bridge;
b) 3 ′ and / or 5 of the nucleoside by dephospho crosslinking
'Replacement of the terminal phosphodiester bridge.

c) replacing the sugar phosphate unit from the sugar phosphate skeleton with another unit;
d) substitution of specific β-D-ribose units with modified sugar units, and e) substitution of natural nucleoside bases with modified nucleoside bases,
Selected from.

  More detailed examples for the chemical modification of oligonucleotides are as follows.

Nucleic acids also include substituted purines and pyrimidines (eg C-5 propyne pyrimidine and 7-
Deaza-7 (substituted purine modified base). Wagner RW et al. (1996) Nat
Biotechnol 14: 840-4. Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2
Aminopurines, 2-amino-6-chloropurines, 2,6-diaminopurines, hypoxanthines, and other naturally and non-naturally occurring nucleoside bases
), Substituted and unsubstituted aromatic moieties. Other such modifications are well known to those skilled in the art. In all of the above embodiments, the X residue is also a non-naturally occurring nucleoside, or nucleotide analog as described herein.

The modified base is any base that is chemically different from the naturally occurring bases normally found in DNA and RNA, such as T, C, G, A, and U, but the basic chemistry with these naturally occurring bases. Share the structure. Modified nucleoside bases are, for example, hypoxanthine, uracil, dihydrouracil, suspect uracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5- (C ₁ -C ₆ ) -alkyluracil, 5- (C _2- C ₆₎ - alkenyl uracil, 5- _(C 2 -C ₆₎ - alkynyl uracil, 5- (hydroxymethyl) uracil, 5-
Chloro uracil, 5-fluorouracil, 5-bromouracil, 5-hydroxy _{cytosine, 5- (C 1 -C 6)} - alkyl _{cytosine, 5- (C 2 -C 6)} - alkenyl cytosine,
5- _(C 2 -C ₆₎ - alkynyl cytosine, 5-chloro-cytosine, 5-fluorocytosine, 5-bromo-cytosine, ^{N 2} - dimethyl guanine, 2,4-diaminopurine, 8-azapurine, substituted 7-deazapurine, Preferably 7-deaza-7-substituted purines and / or 7
-Deaza-8-substituted purines, 5-hydroxymethysine, N4-alkylcytosines such as N4-ethylcytosine, 5-hydroxydeoxycytidine, 5-hydroxymethyldeoxycytidine, N4-alkyldeoxytidine, such as N4-ethyldeoxytidine ,
6-thiodeoxyguanosine, and deoxyribonucleoside of nitropyrrole, C5
-Propynylpyrimidine, as well as diaminopurine (eg 2,6-diaminopurine)
, Inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,
It can be selected from hypoxanthine or other modifications of natural nucleoside bases. This list is meant to be typical and is not to be construed as limiting.

The specific formula described herein defines a set of modified bases. For example, the letter Y is used to refer to nucleosides that contain cytosine or modified cytosine. Modified cytosine, as used herein, is a pyrimidine base analog of naturally occurring or non-naturally occurring cytosine that can replace this base without compromising the immunostimulatory activity of the oligonucleotide. Modified cytosines include, but are not limited to, 5-substituted cytosines (eg,
5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-
Cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-
Cytosine, 5-difluoromethyl-cytosine, and unsubstituted or substituted 5-alkynyl-
Cytosine), 6-substituted cytosine, N4 substituted cytosine (eg, N4-ethyl-cytosine)
, 5-aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudoisocytosine, cytosine analogs having a fused ring system (eg N, N′-propylene cytosine or phenoxazine), and uracil and its derivatives (eg 5 -Fluoro-uracil,
5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil). Some of the preferred cytosines include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodiment of the invention, the cytosine base is a common base (eg, 3-nitropyrrole,
P-base), aromatic ring systems (eg fluorobenzene or difluorobenzene) or hydrogen atoms (dSpacer). The letter Z is used to refer to guanine or a modified guanine base. As used herein, a modified guanine is a naturally occurring or non-naturally occurring purine base analog of guanine that can be substituted for this base without compromising the immunostimulatory activity of the oligonucleotide. Modified guanines include, but are not limited to, 7-deazaguanine, 7-deaza-7-substituted guanine (eg, 7-deaza-7- (C2-C6) alkynylguanine), 7-deaza-8-
Substituted guanine, hypoxanthine, N2-substituted guanine (N2-methyl-guanine), 5
-Amino-3-methyl-3H, 6H-thiazolo [4,5-d] pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenine (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanines (e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. In another embodiment of the invention, the guanine base is a common base (eg, 4-methyl-indole, 5-nitroindole, and K base), an aromatic ring system (eg, benzimidazole or dichloro-benzimidazole). 1-methyl-1H- [1
, 2,4] triazole-3-carboxylic acid amide) or hydrogen atom (dSpacer)
).

Oligonucleotides can include modified internucleotide linkages, such as those described in (a) or (b) above. These modified bonds are partially resistant to degradation (eg, stabilized). A “stabilized nucleic acid molecule” is relatively resistant to in vivo degradation (eg, exonuclease or endonuclease). Stabilization is
It is a function of length or secondary structure. Nucleic acids that are tens to hundreds of kilobases long
Relatively resistant to in vivo degradation. For shorter nucleic acids, secondary structure can stabilize and increase the effect of the nucleic acid. For example, if the 3 ′ end of the nucleic acid has self-complementarity to the upstream region, it will fold to form a kind of stem and loop structure, and then the nucleic acid will begin to stabilize, thereby exhibiting higher activity.

Nucleic acid stabilization can also be achieved through phosphate backbone modifications. Oligonucleotides with phosphorothioate linkages can, in some embodiments, provide maximum activity and protect the oligonucleotide from degradation by intracellular exonucleases and endonucleases.

It has been demonstrated that modification of the nucleic acid backbone enhances the activity of the nucleic acid when administered in vivo. Constructs with phosphorothioate linkages have maximal activity and protect nucleic acids from degradation by intracellular exonucleases and endonucleases. Other modified nucleic acids include phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methylphosphonates, methylphosphorothioates, phosphorodithioates, p-ethoxy, and combinations thereof. These combinations and the specific effects of the combinations on immune cells are described in PCT published patent application PCT / US95 /
01570 (WO 96/022555) and PCT / US97 / 19791 (WO 98)
/ 18810) and US Pat. No. 6,194,388 B1 (issued February 27, 2001), and US Pat. No. 6,239,116 B1 (issued May 29, 2001), C
Discussed against pG nucleic acids. The entire contents of these documents are incorporated herein.

Even if these modified nucleic acids show more stimulating activity due to enhanced nuclease resistance, increased cellular uptake, increased protein binding, increased protein binding, and / or altered intracellular localization It is considered good.

Examples of other stabilized nucleic acids include the following. That is, nonionic DNA analogs such as alkyl and aryl-phosphates (charged phosphonate enzymes are replaced by alkyl or aryl groups), phosphodiesters, and alkylphosphotriates in which the charged oxygen moiety is alkylated. ester. Nucleic acids that are substantially resistant to nuclease digestion also include nucleic acids that contain a diol such as tetraethylene glycol or hexaethylene glycol at one end or the other end.

Oligonucleotides may contain one or two accessible 5 ′ ends. For example, two oligonucleotides can be linked via a 3′-3 ′ bond to
It is possible to generate modified oligonucleotides with two such 5 ′ ends by generating oligonucleotides with one or two accessible 5 ′ ends. 3 '
The -3 'linkage may be a phosphodiester, phosphorothioate, or any other modified internucleoside bridge. Methods for achieving such binding are known in the art. For example, such binding is described in Seliger, H. et al. et al. , Oligonucl
eotide analogs with terminal 3'-3'- and
5'-5'-internucleolytic linkas as antis
ence inhibitors of virtual gene expression
. Nucleosides & Nucleotides (1991), 10 (1-3),
469-77 and Jiang, et al. , Pseudo-cyclic ol
immunoglobulins: in vitro and in vivo prop
erties, Bioorganic & Medicinal Chemistry (
1999), 7 (12), 2727-2735.

In addition, a 3′-3 ′ linked ODN in which the linkage between the 3 ′ terminal nucleosides is not a phosphodiester, phosphorothioate, or other modified bridge is added to an additional spacer (eg, tri-
Or tetra-ethylene glycol phosphate moiety) (Durand, M. et al, Triple-helix formatio).
n by an oligonucleotide containing one (d
A) 12 and two (dT) 12 sequences bridged by
two hexethylene glycol chains, Biochemi
stry (1992), 31 (38), 9197-204, US Pat. No. 5,658,738, and US Pat. No. 5,668,265). Alternatively, non-nucleotide linkers can be obtained from ethanediol, propanediol, or abasic deoxyribose (dSpacer) units (Fontanel, Marie L) using standard phosphoramidite chemistry.
aurence et al. , Sterial recognition by T
4 polynucleotide kinase of non-nucleosid
ic moiets 5'-attached to oligocleoti
des; Nucleic Acids Research (1994), 22 (11),
2022-7). Incorporation of non-nucleotide linkers can be done one or more times or in combination with each other and the two Os to be bound
Any desired distance between the DN 3 ′ ends is possible.

The phosphodiester / nucleotidide bridge located at the 3 ′ and / or 5 ′ end of the nucleoside can be replaced by a modified internucleoside bridge. Here, the modified internucleoside bridge is, for example, phosphorothioate, phosphorodithioate, NR ¹ R ² −
Phosphoramidites, boranophosphates, α-hydroxybenzylphosphonates, phosphate- (C ₁ -C ₂₁ ) -O-alkyl esters, phosphate-[(C ₆ -C ₁₂
) Aryl- (C ₁ -C ₂₁ ) -O-alkyl] ester, (C ₁ -C ₈ ) alkyl phosphonate, and / or (C ₆ -C ₁₂ ) aryl phosphonate bridge, (C ₇ -C ₁₂₎
) -Α-hydroxymethyl-aryl (eg disclosed in WO 95/01363), (C ₆ -C ₁₂ ) aryl, (C ₆ -C ₂₀ ) aryl, and (C ₆ -C ₁₄ )
Aryl is optionally substituted by halogen, alkyl, alkoxy, nitro, cyano, and R ¹ and R ² independently of one another are hydrogen, (C ₁ -C ₁₈ ) alkyl, (C ₆ -C _{2
0) aryl,} (C 6 _{-C 14)} aryl - _(C 1 -C ₈₎ alkyl, preferably hydrogen,
(C ₁ -C ₈ ) alkyl, preferably (C ₁ -C ₄ ) alkyl, and / or methoxyethyl, or R ¹ and R ² together with the nitrogen atom bearing them, from the group of O, S, and N To form a 5-6 membered heterocyclic ring which may further comprise

Dephospho bridge (for example, Uhlmann E and Pe
yman A in "Methods in Molecular Biology"
, Vol. 20, “Protocols for Oligonucleotides”
es and Analogs ", S.A. Agrawal, Ed. , Humana
Press, Totowa 1993, Chapter 16, pp. 355
substitution of phosphodiesters located at the 3 ′ and / or 5 ′ end of the nucleoside, as described in ff), dephosphobridges, for example, dephosphobridged formacetal (formac
etal), 3′-thioformacetal, methylhydroxylamine, oxime, methylenedimethylhydrazo, dimethylenesulfone, and / or silyl group.

The composition of the present invention may optionally have a chimeric skeleton. As used herein, a chimeric backbone is a backbone that includes two or more types of bonds. In one embodiment, the chimeric backbone can be represented by the formula: 5′Y ₁ N ₁ ZN ₂ Y ₂ 3 ′. Y ₁ and Y ₂ are nucleic acid molecules having 1 to 10 nucleotides. Y ₁ and / or Y ₂ each contain at least one modified internucleotide linkage. Since at least two nucleotides of the chimeric oligonucleotide contain backbone modifications, these nucleic acids are an example of one type of “stabilized immunostimulatory nucleic acids”.

For chimeric oligonucleotides, Y ₁ and Y ₂ are considered independent of each other. This means that each of Y ₁ and Y ₂ may or may not have different sequences and different skeletal bonds within the same molecule. In some embodiments, Y ₁ and Y ₂ have 3-8 nucleotides. N ₁ and N ₂ are nucleic acid molecules having 0-5 nucleotides as long as N ₁ ZN ₂ has a total of at least 6 nucleotides. The nucleotide of N ₁ ZN ₂ has a phosphodiester backbone and does not include nucleic acids with a modified backbone. Z is an immunostimulatory nucleic acid motif and is preferably selected from those shown herein.

The central nucleotide (N ₁ ZN ₂ ) of formula Y ₁ N ₁ ZN ₂ Y ₂ has a phosphodiester internucleotide linkage, and Y ₁ and Y ₂ have at least one modified internucleotide linkage, There may be more than one or all modified internucleotide linkages. In preferred embodiments, Y ₁ and / or Y ₂ has at least two, or two to five modified internucleotide linkages, Y ₁ has two modified internucleotide linkages, and Y ₂ is
Either have 5 modified internucleotide linkages, or Y ₁ has 5 modified internucleotide linkages, and Y ₂ has 2 modified internucleotide linkages. The modified internucleotide linkage is, in some embodiments, a phosphorothioate modified linkage, a phosphorodithioate modified linkage, or a p-ethoxy modified linkage.

The nucleic acid also includes a nucleic acid having a backbone sugar, and the backbone sugar is covalently bonded at a 2 ′ position to a low molecular weight organic group other than a hydroxyl group and at a 5 ′ position to a low molecular weight organic group other than a phosphate group. Thus, the modified nucleic acid may contain a 2′-O-alkylated ribose group. Furthermore, the modified nucleic acid may contain sugars such as arabinose or 2′-fluoroarabinose instead of ribose. Thus, since nucleic acids can be heterogeneous in backbone composition, they contain any possible combination of polymer units that bind to each other, such as peptide-nucleic acids (having an amino acid backbone with a nucleobase). In some embodiments, the nucleic acids are homologous in the backbone composition. Other examples are described in more detail below.

Sugar phosphate units derived from sugar phosphate skeletons (that is, sugar phosphate skeletons are composed of sugar phosphate units) (that is, β-D-ribose and phosphate diesters collectively forming sugar phosphate units) Internucleoside bridge) can be replaced with another unit,
At this time, the other unit is, for example, “morpholino-d”
erivative) ”oligomers (eg Stirchak EP et
al. (1989) Nucleic Acids Res 17: 6129-41.
(Ie, for example, substitution with a morpholino derivative unit),
Or construct a polyamide nucleic acid (“PNA”) (eg, Nielsen PE et
al. (1994) Bioconjug Chem 5: 3-7), i.e. suitable for substitution, e.g. with 2-aminoethylglycine, e.g. with a PNA backbone unit. Oligonucleotides have other carbohydrate backbone modifications and substitutions (eg, peptide nucleic acids having phosphate groups (PHONA), locked nucleic acids (LNA), and oligonucleotides having backbone portions with certain alkyl or amino linkers) May be included. The alkyl linker may be branched or unbranched, may be substituted or unsubstituted, and may be chirally pure or a racemic mixture.

The β-ribose unit or β-D-2′-deoxyribose unit can be replaced with a modified sugar unit, wherein the modified sugar unit is, for example, β-D-ribose, α-D-2 '- deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-F- _{arabinose, 2'-O- (C 1 -C} 6) alkyl - ribose (preferably, _{2'-O- (C 1 -C 6} ) alkyl - ribose is 2'-O- methyl _{ribose), 2'-O- (C 2} -C 6) alkenyl - ribose, 2 '- [O- ( C ₁ −
C ₆₎ alkyl -O- _(C 1 -C ₆₎ alkyl] - ribose, _2'-NH 2-2'-deoxyribose, beta-D-xylo - furanose, alpha-arabinofuranose, 2,4-dideoxy -Β-D-erythro-hexo-pyranose, as well as carbocyclic sugar analogs (eg
Froehler J (1992) Am Chem Soc 114: 8320) and / or acyclic sugar analogs (e.g., Vandendriesche e
t al. (1993) Tetrahedron 49: 7223) and / or
Or a bicyclosaccharide analogue (eg, Tarkov M et al. (1993) He
lv Chim Acta 76: 481).

In some embodiments, the sugar is 2'-O-methyl ribose, particularly for one or both nucleotides joined by a phosphodiester or phosphodiester-like internucleoside linkage.

For use in the present invention, the oligonucleotides of the present invention can be synthesized de novo using any of a number of methods well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage, SL, and Caruthers
, M.M. H. , Tet. Let. 22: 1859, 1981), nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27: 4051-40405, 1
986; Froehler et al. , Nucl. Acid. Res. 14: 539
9-5407, 1986; Garegg et al. , Tet. Let. 27:40
55-4058, 1986, Gaffney et al. , Tet. Let. 29: 2
619-2622, 1988). By various automatic nucleic acid synthesizers that are commercially available,
These chemistries can be performed. These oligonucleotides are referred to as synthetic oligonucleotides. Alternatively, T-rich and / or TG dinucleotides are produced in large quantities in plasmids (Sambrook, T., et al., “Molecula
r Cloning: A Laboratory Manual ", Cold Spr
ing Harbor laboratory Press, New York, 198
9)) can be separated into smaller pieces or administered as a whole. Nucleic acids can be prepared from existing nucleic acid sequences (eg, genomic DNA or cDNA) using known techniques, such as those using restriction enzymes, exonucleases, or endonucleases.

Modified backbones such as phosphorothioates can be synthesized using automated techniques utilizing either phosphoramidite or H-phosphonate chemistry. For example, US Pat.
469,863, aryl-phosphonates and alkyl-phosphonates can be produced. Alkyl phosphotriesters (US Pat. No. 5,023,243 and European Patent No. 092,57) were prepared by automated solid-phase synthesis using commercially available reagents.
As described in No. 4, the charged oxygen moiety is alkylated). Methods for generating other DNA backbone modifications and substitutions have been described (eg, U
hlmann, E .; and Peyman, A .; , Chem. Rev. 90: 544,1
990; Goodchild, J .; , Bioconjugate Chem. 1: 165
1990).

Nucleic acids prepared in this way are referred to as isolated nucleic acids. In general, an “isolated nucleic acid” refers to a nucleic acid that has been separated from the components with which the nucleic acid is normally associated in nature. In one example, the isolated nucleic acid is from a cell, from a nucleus,
It can be isolated from mitochondria or from chromatin.

When administering a nucleic acid in combination with an antigen encoded in a nucleic acid vector (as described herein), the backbone of the nucleic acid is a phosphodiester and phosphorothioate (or other phosphate ester modification) chimera. Preferred combinations are preferred. Cells may have trouble taking up plasmid vectors in the presence of intact phosphorothioate nucleic acids. Thus, when delivering both a vector and nucleic acid to a subject, the nucleic acid preferably has a chimeric backbone or a phosphorothioate backbone, but associates the plasmid with a carrier that delivers the plasmid directly to the cell. Thus, it is preferable to eliminate the need for intracellular uptake. Such carriers are known in the art and include, for example, liposomes and gene guns.

The present invention further encompasses the use of any of these nucleic acids described above in the methods recited herein and all of the use of immunostimulatory nucleic acids as previously described and known. To do.

It has been discovered by the present invention that the immunostimulatory nucleic acid surprisingly increases the immunostimulatory effect. For example, it has been demonstrated that the nucleic acids described herein can provide protection against infection, perhaps by largely stimulating the immune system. In an example, a mouse subject challenged with herpes simplex virus type 2 (HSV-2) of a nucleic acid having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 19, SEQ ID NO: 45, SEQ ID NO: 118, or SEQ ID NO: 141 Illustrate ability to protect. The nucleic acid can be administered prior to or simultaneously with the viral attack.

The ability of these nucleic acids to be demonstrated to induce immunostimulation is such that they are vaccinated, cancer immunotherapy, asthma immunotherapy, and overall enhancement of immune function in humans and other subjects,
Evidence that it is an effective therapeutic agent for enhanced hematopoietic recovery after radiation or chemotherapy and other immunomodulatory treatments.

The nucleic acids of the invention can be used as stand-alone therapies. Stand-alone therapy is a therapy that can achieve prophylactically or therapeutically useful results from administration of a single drug or composition. Thus, the nucleic acids described herein can be used alone in the prevention or treatment of infectious diseases, cancer, and asthma and allergies. This is because the nucleic acid can induce an immune response useful for the therapeutic outcome of these diseases. Some of the methods described herein relate to the use of nucleic acids as stand-alone therapies, while others
Related to the use of nucleic acids in combination with other therapeutic agents.

When used in a vaccine, the nucleic acid is administered with an antigen. Preferably, the antigen is
Specific to disorders that require prevention or treatment. For example, if the disorder is an infectious disease, the antigen is preferably derived from an infectious organism (eg, bacteria, viruses, parasites, fungi, etc.).
Where the disorder is cancer, the antigen is preferably a cancer antigen.

Immunostimulatory nucleic acids are useful in some aspects of the invention as prophylactic vaccines for the prevention of infection (ie, infection), cancer, allergy, or asthma. Preferably, a subject who has not been diagnosed as suffering from one of these symptoms, more preferably a subject who is considered at risk of developing one of these symptoms, is prophylactic vaccination Is used. For example, the subject is at risk of developing an infection with an infectious organism, is at risk of developing a cancer for which a specific cancer antigen is identified, or develops an allergy with a known allergen It may be at risk of developing asthma or a predisposition to developing asthma.

As used herein, a subject at risk is a subject who has some risk of exposure to an infectious agent, carcinogen, or allergen. A subject at risk also includes a subject having a predisposition to developing such a disorder. Some predispositions can be hereditary (thus identified by either genetic analysis or family history). Some predispositions are environmental (eg, prior exposure to carcinogens). Examples of subjects at risk of developing an infection include subjects or infectious organisms where a particular type of infectious agent is detected, or where they live or are expected to travel It can be a subject that is directly or indirectly exposed to an organism through lifestyle or medical treatment by contact with a bodily fluid that it may contain. Subjects at risk of developing an infection also include a general population recommended by a medical institution to vaccinate against a particular infectious organism.

If the antigen is an allergen and the subject develops an allergic response to that particular antigen and the subject may be exposed to the antigen (ie, in the pollen season), the subject There is a risk of exposure. Subjects at risk of developing asthma allergies have been identified as having allergies or asthma but no active disease during immunostimulatory nucleic acid treatment, and genetic or environmental And subjects considered to be at risk of developing these diseases due to factors.

Immunostimulatory nucleic acids are for short-term protection against infections, allergies, or cancer
It can also be given without antigens or allergens, in which case repeated doses allow longer term protection.

A subject at risk of developing cancer is one who is more likely to develop cancer (eg, more likely than in the general public). These subjects include, for example, subjects with genetic abnormalities (the presence of the genetic abnormalities has been demonstrated to correlate with a higher likelihood of developing cancer than in the general public) A subject who has been exposed to a cancer-causing factor (ie, a carcinogen), such as tobacco, asbestos, or other chemical toxins, or has previously treated cancer,
And subjects who are in remission on the surface. A subject at risk of developing cancer
When the subject is treated with an antigen specific for the type of cancer at risk of developing and an immunostimulatory nucleic acid, the subject kills the cancer cell as it develops May be possible. When a tumor begins to form in a subject, the subject generates a specific immune response against the tumor antigen.

In addition to using immunostimulatory nucleic acids as a preventive measure, the present invention provides infectious diseases, allergies,
Also encompassing the use of immunostimulatory nucleic acids for the treatment of subjects suffering from asthma or cancer.

A subject suffering from an infectious disease is exposed to an infectious agent and in the body or excreta,
A subject having an acute or chronic detectable level of a pathogen. When used therapeutically, the immunostimulatory nucleic acid can be used as a stand alone or in combination with another therapeutic agent. For example, immunostimulatory nucleic acids can be used therapeutically with antigens that increase the level of antigen-specific systemic or mucosal immune responses that can reduce the level of infectious pathogens or eliminate infectious pathogens.

As used herein, an infectious disease is a disease caused by the presence of foreign microorganisms in the body. It is particularly important to develop effective vaccine strategies and treatments that protect the mucosal surface of the body, which is the main pathogen entry site.

Treat as used herein with respect to infections,
The term treated or treating refers to a prophylactic treatment that increases the resistance of a subject (subject at risk of infection) to infection with a pathogen, or another In other words, a subject (infectious) to prevent a prophylactic treatment that reduces the likelihood that the subject will be infected with a pathogen and to fight the infection (eg, reduce or eliminate or prevent the infection from worsening). Treatment after the subject is infected.

The subject suffering from an allergy has an allergic reaction in response to an allergen,
Or a subject at risk of developing an allergic reaction. Allergy refers to acquired hypersensitivity to a substance (allergen). Allergic symptoms include, but are not limited to, eczema, allergic rhinitis or coryza, hay fever, conjunctivitis, bronchial asthma, urticaria and food allergies, and other atopic symptoms.

Currently, allergic diseases are generally treated by injection of small amounts of antigen followed by increasing doses of antigen. This method is thought to induce tolerance to allergens and prevent further allergic reactions. However, these methods can take years to become effective and are associated with the risk of side effects such as anaphylactic shock. The method of the present invention avoids these problems.

Allergies are generally caused by IgE antibody production against harmless allergens. Cytokines induced by systemic or mucosal administration of immunostimulatory nucleic acids are, for the most part, a class of cytokines called Th1 (examples include IL-12 and IFN-γ), which are humoral and Induces both cellular immune responses. The type of antibody associated with the Th1 response is generally more protective because it has a high neutralizing and opsonizing ability. Other major types of immune responses are IL-4, IL-5, and IL-10
It is associated with cytokine production and is referred to as the Th2 immune response. The Th2 response is mostly accompanied by antibodies and has a less protective effect against infection, with several Th2 isotypes (
For example, IgE) is associated with allergies. In general, allergic diseases are mediated by a Th2-type immune response, while excessive Th1 responses are associated with autoimmune diseases, while Th
One response seems to give the best protection against infection. Based on the ability of the immunostimulatory nucleic acid to transfer the subject's immune response from a Th2 (related to the generation of IgE antibodies and allergies) to a Th1 response (protective against allergic reactions), A dosage effective to induce an immune response can be administered to a subject to treat or prevent allergies.

Thus, immunostimulatory nucleic acids have significant therapeutic utility in the treatment of allergic and non-allergic conditions such as asthma. Th2 cytokines, particularly IL-4 and IL-5, are increased in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response including IgE isotope switching, neutrophil migration and activation, and mast cell proliferation. Th1 cytokines, particularly IFN-γ and IL-12, can suppress the formation of Th2 clones and the production of Th2 cytokines. Asthma refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways, and increased airway responsiveness to inhaled factors. Asthma is often associated with, but not limited to, atopic or allergic symptoms.

A subject suffering from cancer is a subject having detectable cancer cells. The cancer can be malignant or non-malignant cancer. Cancers or tumors include but are not limited to biliary tract cancer, brain cancer, breast cancer,
Cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, carcinoma in situ, lymphoma, liver cancer, lung cancer (eg small cells and non-small cells), melanoma, neuroblastoma, oral cavity Examples include cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, thyroid cancer, and renal cancer, and other carcinomas and sarcomas. In one embodiment, the cancer is ciliary cell leukemia, chronic myelogenous leukemia, cutaneous T cell leukemia, multiple myeloma, follicular lymphoma, malignant melanoma, squamous cell carcinoma, renal cell carcinoma, prostate carcinoma, bladder Cell carcinoma or colon carcinoma.

Some cancer cells are antigenic and can therefore be targets of the immune system. In one aspect, administration of immunostimulatory nucleic acids in combination with anti-cancer agents, particularly those classified as cancer immunotherapy, is useful for stimulating specific immune responses against cancer antigens.

The theory of immune surveillance is that the primary function of the immune system is to detect and remove neoplastic cells before tumor formation. The basic principle of this theory is that cancer cells are antigenically different from normal cells,
It induces an immune response similar to that which causes rejection of immunologically incompatible allografts. Studies have confirmed that tumor cells differ in their antigen expression qualitatively or quantitatively. Such antigens are interchangeably referred to as tumor antigens or cancer antigens. Some of these antigens can then be tumor-specific antigens or tumor-associated antigens. "Tumor-specific antigen (tumor-specific antigen)
n) ”is an antigen that is specifically present in tumor cells but not in normal cells. Examples of major specific antigens are viral antigens in tumors induced by DNA or RNA viruses. “Tumor-associated” antigens are present in both tumor cells and normal cells, but are present in different amounts or in different forms in tumor cells.
Examples of such antigens include oncofetal antigens (eg, carcinoembryonic antigen), differentiation antigens (eg,
T antigen and Tn antigen) and oncogene products (eg HER / neu).

Various types of cells have been identified that can kill tumor targets in vitro and in vivo: natural killer cells (NK cells), cytolytic T lymphocytes (CTL), lymphokine activated killer cells (LAK) , And activated macrophages. NK cells can kill tumor cells without prior sensitization to specific antigens, and their activity is a class I antigen encoded by the major histocompatibility complex (MHC) on target cells Does not require the presence of. NK cells are thought to be involved in the control of neoplastic tumors and the control of metastatic growth. In contrast to NK cells, CTL can kill tumor cells after being sensitized to tumor antigens and only when the target antigen is expressed on tumor cells that also express MHC class I. . CTLs are thought to be effector cells in tumor rejection due to transplanted tumors and DNA viruses. LAK cells are a subset of null lymphocytes that are distinct from the NK and CTL populations. Once activated, macrophages are capable of killing tumor cells in a manner that is not antigen dependent and not limited to MCH. Activated macrophages are thought to reduce the growth rate of tumors infiltrated by them. In vitro assays have identified other immune mechanisms such as antibody-dependent cell-mediated cytotoxic responses and lysis by antibodies and complement. However, these immune effector mechanisms
It is thought to be less important in vivo than NK, CTL, LAK, and macrophage functions in vivo (for review see Piessens, WF, and Da
vid, J.M. , “Tumor Immunology”, Scientific Ame
rican Medicine, Vol. 2, Scientific American B
ooks, N.M. Y. , Pages 1-13, 1996).

The goal of immunotherapy is to increase the patient's immune response to established tumors. One method of immunotherapy includes the use of adjuvants. Adjuvant substances derived from microorganisms such as Bacillus Calmette-Guerin enhance the immune response and enhance tumor resistance in animals.

As used herein, an “antigen” is a molecule that can elicit an immune response. Antigens include, but are not limited to, cells, cell extracts, proteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, polysaccharides and peptide and non-peptidomimetics of other molecules, small molecules, lipids, glycolipids, sugars Quality, viruses and virus extracts, and multicellular organisms such as parasites and allergens. The term antigen broadly includes any type of molecule that is recognized as being foreign to the host immune system. Antigens include, but are not limited to, cancer antigens, microbial antigens, and allergens.

As used herein, a “microbial antigen” is a microbial antigen, including but not limited to viruses, bacteria, parasites, and fungi. Such antigens include intact microorganisms and their natural isolates and fragments or derivatives, which are identical or similar to natural microbial antigens and induce an immune response specific for that microorganism. Also included are synthetic compounds. A compound is similar to a natural microbial antigen when it induces an immune response (humoral and / or cellular) to the natural microbial antigen. Such antigens are routinely used in the art and are well known to those skilled in the art.

As used herein, a “cancer antigen” is a compound, such as a peptide or protein, present on a tumor or cancer cell that is expressed on the surface of antigen-presenting cells in the context of MHC molecules. A compound that can elicit an immune response. Prepare a crude extract of cancer cells (see, eg, Cohen et al., 1994, Cancer Re
cancer, as described in search, 54: 1055), by partially purifying the antigen, by recombinant techniques, or by novel synthesis of known antigens. it can. Cancer antigens include, but are not limited to, antigens that are recombinantly expressed, an immunogenic portion of a tumor or cancer, or an entire tumor or cancer. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.

By differentially expressing cancer or tumor antigens by cancer cells, the cancer or tumor antigens can be utilized to target cancer cells. Some of these antigens are not necessarily expressed, but are encoded by normal cells. Characterize these antigens as being normally silent (ie not expressed) in normal cells, expressed only at certain stages of differentiation, and transiently expressed, such as embryonic and fetal antigens be able to. Other cancer antigens include oncogenes (eg, activated ras oncogene)
, Encoded by mutant cell genes such as suppressor genes (eg, mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations. Still other cancer antigens include RNA and D
It can be encoded by viral genes such as those carried on NA tumor viruses.

In some aspects of the invention, the subject is exposed to the antigen “exposed to
) " As used herein, the term “exposed to” refers to either an active step of contacting a subject with an antigen or passively exposing a subject to an antigen in vivo. Point to. Methods for actively exposing a subject to an antigen are well known in the art. In general, the antigen is administered directly to the subject by any means such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration.
The antigen can be administered systemically or locally. Methods for administering antigens and immunostimulatory nucleic acids are described in more detail below. If the antigen is available for exposure to immune cells in the body, the subject is passively exposed to the antigen. For example, a subject can be passively exposed to an antigen by invading a foreign pathogen into the body or by generating tumor cells that express a foreign antigen on the cell surface.

The method of passively exposing a subject to an antigen can depend in particular on the timing of administration of the immunostimulatory nucleic acid. For example, in a subject at risk of developing a cancer or infection or an allergic or asthmatic response, the subject is at the maximum risk, i.e. during the allergy season or after exposure to a causative agent. Immunostimulatory nucleic acids can be administered periodically. In addition, immunostimulatory nucleic acids can be administered to travelers prior to traveling to a foreign country at risk of exposure to infectious agents. Similarly, if immunostimulatory nucleic acids are administered to soldiers or citizens at risk of being exposed to biological warfare, and if the subject is exposed to or is exposed to biological warfare, systemic or mucosal to antigens An immune response can be induced.

In some preferred embodiments, administration is local, but nucleic acids and other therapeutic agents can be administered systemically. Topical administration may include topical application to mucosal surfaces such as the mucosal surface of the mouth, vagina, anus, and penis. In embodiments where administration is local, particularly to the vaginal, anal and mucosal surfaces of the mouth, the nucleic acid is preferably other than a CpG nucleic acid.

In certain embodiments, the present invention uses local mucosal administration of unmethylated CpG nucleic acid to
IV-1, HIV-2, HIV-3, HTLV-I, HTLV-II, HTLV-III
, Hepatitis A virus, hepatitis B virus, herpes simplex virus (HSV) type 1 and type 2,
Papillomavirus, Neisseria gonorrhoeae, Treponem
a pallidum, Campylobacter sp. , Cytomegalovirus (
CMV), Chlamydia trachomatis, and Candida a
It is intended to prevent or treat human sexually transmitted diseases (STD) caused by lbicans.

As used herein, STD is an infection that is transmitted primarily through sexual intercourse, without limitation. In addition to being transmitted via sexual contact with an infected subject, some S
TD can also be transmitted through contact with body fluids of infected subjects. As used herein, “body fluid” includes blood, saliva, semen, vaginal fluid, urine, stool, and tears. STD is most commonly transmitted via blood, saliva, semen, and vaginal fluid. As an example, blood and blood product transfusions are a common mode of transmission for many sexually transmitted pathogens including HIV and hepatitis viruses.

Sexual pathogens are generally bacterial, viral, parasitic or fungal in nature. Examples of organisms that cause STD include Neisseria gonorrhoeae, C
hlamydia trachomatis, Treponema pallidum,
Haemophilus ducreyi, Condyloma acminata,
Calymmatobacterium granulomatis, and Ureap
bacteria such as lasma urealyticum and human immunodeficiency virus (HIV-1)
And HIV-2), human T lymphotropic virus type I (HTLV-I), herpes simplex virus type 2 (HSV-2), human papilloma virus (plural types), hepatitis B virus, cytomegalovirus, and infection Viruses such as genital molluscum virus and Trichomona
parasites such as s vaginalis and Phthirus pubis,
and fungi such as da albicans.

Other infections are known to be transmitted sexually even though sexual transmission is not their primary mode of transmission. This latter category includes Mycoplasma homini
bacteria such as S, Gardnerella vaginalis, and group B streptococci, and human T lymphotropic virus type II (HTLV-II), hepatitis C and D viruses, herpes simplex virus type I (HSV-1) and Epstein Bar Infectious diseases caused by viruses such as viruses (EBV) and parasites such as Sarcoptes scabiei.

The present invention is also intended to encompass STDs that are transmitted by sexual contact, including oral and fecal exposure. These STDs are available from Shigella spp. And Campyloba
cter spp. Such as bacteria, viruses such as hepatitis A, and Giardia lambl
Caused by parasites such as ia and Entamoeba histolytica.

A “subject in need thereof” is a subject at risk of developing STD or one having STD (ie, STD
A subject suffering from D).

Nucleic acids are useful in several aspects as a preventive measure to prevent STD in a subject at risk of developing STD. As used herein, “subject at risk of developing an STD”.
“D)” is a subject who has some risk of developing STD, either by contact with an infected subject or by contact with a body fluid from an infected subject. For example, a subject at risk is one who currently has or will have a sexual partner who is infected with an STD causative agent. Risky subjects may also use the condom (ie, male or female condom), regardless of whether they are or are aware of an existing infection, Or those involved in unprotected sexual acts such as having sexual intercourse in either the vagina. Subjects with multiple sexual partners (eg,
Prostitutes or those who are prostituted frequently) or even subjects with one sexual partner who has multiple sexual partners are considered at risk. Other subjects at risk of developing STD are those involved in other forms of high-risk infectious behavior, such as sharing a hypodermic needle. A subject receiving a blood product may also be considered at risk, especially if the blood supply system is not strictly monitored. Examples of this latter category of subjects are subjects in sub-Saharan African countries with blood supply systems that are partially or completely contaminated with STD-causing pathogens (eg, HIV). A subject at risk is one in which one or more STD-causing pathogens are planning a trip to a common area (particularly known to be present in the blood supply system of the area) It can be). Another subject at risk is one that has an occupation that includes potential contact with another person's body fluids. Examples of this latter category include, but are not limited to, nurses, doctors, dentists, and emergency personnel (eg, ambulance personnel, emergency medical personnel, firefighters, and police officers). At risk subjects also include fetuses and newborns born to mothers infected with STD-causing pathogens.

All of the aforementioned activities related to the transmission of STD causative pathogens are "high risk activities (hig
h risk activity). Nucleic acids and other potential prophylactic or therapeutic agents used in combination can be administered before, during, or after the subject's involvement in high-risk activity. Subjects who are administered nucleic acids prior to engaging in sexual activity can have, for example, at least 1 month, at least 1 week, at least 4 weeks prior to having sexual intercourse
8 hours, at least 24 hours, at least 12 hours, at least 6 hours, at least 4 hours, at least 2 hours (or any time between the times explicitly listed herein)
Can receive nucleic acids. Preferably, the time of administration prior to participating in the high-risk activity is sufficient to activate the immune system so that it is active while the infectious agent is present in the subject's body. Subjects who receive nucleic acid after being involved in high-risk activity are within 2 hours, within 4 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours after participating in high-risk activities Nucleic acids can be received within 3, 4, 5, 6, 7, 14, 28 days or longer (or any time between the times explicitly listed herein).

Preferably, the subject is a non-rodent subject. Non-rodent subjects include, but are not limited to, dogs, cats, horses, cows, pigs, sheep, goats, chickens, primates (eg monkeys), fish (aquaculture species such as salmon), And in particular humans or vertebrates excluding rodents such as rats and mice.

Antigens can be derived from a variety of sources including tumors, non-tumor cancers, allergens, infectious agents. Each of the lists listed herein is not intended to be limiting.

Examples of viruses found in humans include, but are not limited to, Retrovirida
e (eg, human immunodeficiency virus such as HIV-1 (also referred to as HTLV-III, LAV, or HTLV-III / LAV, or HIV-III) and HIV-L
Other isolates such as P, Picoraviridae (eg, poliovirus, hepatitis A virus, enterovirus, human coxsackie virus, rhinovirus, echovirus) and calciridae (eg, strains causing gastroenteritis) ) And T
ogaviridae (eg, equine encephalitis virus, rubella virus) and Flaviri
dae (eg, dengue virus, encephalitis virus, yellow fever virus) and Coronov
iridae (eg, coronavirus), Rhabdoviradae (eg, vesicular stomatitis virus, rabies virus), Coronaviridae (eg, coronavirus), Rhabdoviradae (eg, vesicular stomatitis virus, rabies virus), and Filobirida (eg, , Ebola virus) and Paramyxov
iriidae (eg, parainfluenza virus, mumps virus, measles virus, respiratory rash virus), Orthomyxoviridae (eg, influenza virus), and Bunyaviridae (eg, huntan virus, bunya virus, flevovirus, and Nairo virus) and Arena viri
dae (haemorrhagic fever virus), Reoviridae (eg, reovirus, orbivirus, and rotavirus), Birnaviridae, and Hepadnavir
idae (hepatitis B virus), Parvovirida (parvovirus) and P
apoviridae (papilloma virus, polyoma virus) and Ade
noviridae (most adenoviruses) and Herpesviridae (herpes simplex (HSV) types 1 and 2, varicella-zoster virus, cytomegalovirus (C
MV), herpes virus) and Poxviridae (decubitus virus, vaccinia virus)
Viruses, pox viruses), Iridoviridae (eg, African swine fever virus), and unclassified viruses (eg, causative factors of spongiform encephalopathy, factors of hepatitis delta (deficient satellite of hepatitis B) ), Non-A and non-B hepatitis factors (class 1 is internally transmitted, class 2 is parenteral (ie, hepatitis C)), Norwalk and related viruses, and astrovirus) Is mentioned.

Although many of the microbial antigens have been described herein in connection with human disease, the present invention
It is also useful for treating other non-human diseases. Non-human vertebrates can also develop infections that can be prevented or treated with the immunostimulatory nucleic acids described herein. For example, in addition to treating infectious human diseases, the methods of the present invention are useful for treating animal infections.

Both gram negative and gram positive bacteria function as antigens in vertebrates. Such Gram-positive bacteria include, but are not limited to, Pasteurella species, Staph
ylococci species, and Streptococcus species. Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomo.
Examples include nas species and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pyloris, Borelia b
urgdorferi, Legionella pneumophilia, Mycob
acteria sps (eg, M. tuberculosis, M. avium, M
. intracellulare, M. et al. Kansaii, M.M. gordonae), St
aphylococcus aureus, Neisseria gonorrhoea
e, Neisseria meningitidis, Listeria ruonoc
ytogenes, Streptococcus pyogenes (Group A Streptococcus),
Streptococcus agalactiae (Group B Streptococcus), Strepto
coccus (viridans group), Streptococcus faecalis
, Streptococcus bovis, Streptococcus (anaer
obic species), Streptococcus pneumoniae, pathogenic Campy
lobacter sp. Enterococcus sp. , Haemophilu
s influenzae, Bacillus anthracis, corynebac
terium diphtheriae, corynebacterium sp. , E
rysiperothrix rhusiopathiae, Clostridium
perfringers, Clostridium tetani, Enterobac
ter aerogenes, Klebsiella pneumoniae, Past
urella multicida, Bacteroides sp. , Fusobac
terium nucleatum, Streptobacillus monif
ormis, Treponema pallidium, Treponema part
enue, Leptospira, Rickettsia, and Actinomyce
s israelli.

Bacterial pathogen polypeptides include, but are not limited to, iron-regulated outer membrane protein (IRROM), outer membrane protein (OMP), and Aeromoni that cause Frunker's disease
s salmonicida A protein and R causing bacterial kidney disease (BKD)
the p57 protein of Enibacterium and the major surface associated antigens of Yersinia (m
ajor surface associated antigen (msa) and surface-expressed cytotoxin (mpr)
), Surface expressed hemolysin (is
h)), and flagellar antigen, an extracellular protein (ECP), iron-regulated outer membrane protein (IRROM), and a structural protein, and Vibrosis angle
arum and V.A. ordalii's OMP and flagellar proteins and Edwards
Iellosis ictaluri and E. coli. tarda flagellar protein, OMP protein, aroA, and purA, Ichthyophthirius surface antigen, Cytophaga columnari structure and regulatory protein, Rick
and the structure and regulatory proteins of ettsia.

Parasite pathogen polypeptides include, but are not limited to, Ichthyophth
The surface antigen of irius is mentioned.

Examples of fungi include Cryptococcus neoformans, Histo
plasma capsulatum, Coccidioides immitis, B
lastmyces dermatitidis, Chlamydia tracho
matis, Candida albicans. Other infectious organisms (ie, protists) include Plasmodium falciparum, Plasmo
dium malariae, Plasmodium ovale, and Plasmo
Plasmodium species such as dime vivax and Toxoplasma gond
ii. As blood-derived and / or tissue parasites, Plasmodi
m species, Babesia microti, Babesia divergens, Lei
shmania tropica, Leishmania species, Leishmania b
razilianis, Leishmania donovani, Trypanos
oma gambiense, and Trypanosoma rhodesense
(African sleep disease), Trypanosoma cruzi (Chagas disease), and T
and oxoplasma gondii. Other medically relevant microorganisms are extensively described in the literature. For example, C.I. G. A Thomas, Medica
l Microbiology, Bailier Tindall, Great B
Reference is made to ritain 1983, the entire contents of which are incorporated herein.

For many vaccines for the treatment of non-human vertebrates, see Bennett, K. et al. Comp
endium of Veterinary Products, 3rd ed. Nor
th American Compensium, Inc. , 1995. As mentioned above, antigens include microorganisms such as viruses, parasites, bacteria, and fungi derived from natural sources or synthesized, and fragments thereof.
Human and non-human vertebrate infectious viruses include retroviruses, RNA viruses, and DNA viruses. This group of retroviruses includes both single retroviruses and complex viruses. Single retroviruses include subgroups of type B retroviruses, type C retroviruses, and type D retroviruses. An example of a type B retrovirus is mouse mammary tumor virus (MMTV). Type C retroviruses include group C group A (Rous sarcoma virus (RSV), avian leukemia virus (
ALV) and avian myeloblastosis virus (AMV)) and group C (feline leukemia virus (FeLV), gibbon leukemia (GALV), spleen necrosis virus (SNV),
Subgroup with reticuloendotheliosis virus (RV) and simian sarcoma virus (SSV). Type D retroviruses include Mason-Pfizer Monkey virus (MPMV) and simian retrovirus type 1 (SRV-1). Complex retroviruses include lentivirus, T cell leukemia virus, and foamy
A subgroup of viruses. Lentiviruses include HIV-1, but also include HIV-2, SIV, visna virus, feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV). As T cell leukemia virus, H
TLV-1, HTLV-II, monkey T cell leukemia virus (STLV), and bovine leukemia virus (BLV). Foamy viruses include human foamy virus (HFV), simian foamy virus (SFV), and bovine foamy virus (BFV).

Examples of other RNA viruses that are antigens in vertebrates include, but are not limited to, Ort
horeovirus (multiple serotypes of both mammalian and avian retroviruses), O
genus rbirrus (bluetongue virus, Eugenangele virus, kemerovo virus, African equine disease virus, and Colorado tick fever virus), Rot
Reovirida including genus avirus (human rotavirus, Nebraska calf diarrhea virus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus)
e family members and Enterovirus (Poliovirus, Coxsackie virus type A and B, enterocyte necrotic human orphan (ECHO) virus, hepatitis A virus, simian enterovirus, mouse encephalomyelitis (ME) virus, Poliovirus muris, bovine enterovirus, porcine enterovirus, Cardiovirus genus (encephalomyocarditis virus (EMC), Mengo virus), Rhinovirus genus (human rhinovirus containing at least 113 subtypes), etc. Rhinovirus), P containing the genus Apthovirus (FMDV)
Ca containing the icornaviridae family and swine vesicular rash virus, San Miguel seal virus, feline picornavirus, and Norwalk virus
lciviridae and Alphavirus genus (Eastern equine encephalitis virus, Semliki Forest virus, Sindbis virus, Chikungunya virus, Onyonyon virus, Ross River virus, Venezuela equine encephalitis virus, Western equine encephalitis virus)
, Flavirius genus (mosquito-borne yellow fever virus, dengue virus, Japanese encephalitis virus,
St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Kunjin virus, Central European tick-borne virus, Far Eastern tick-borne virus, Kyasanua Forest virus, Jumping disease virus, Poissan virus, Omsk hemorrhagic fever virus),
Togaviridae family including Rubivirus (rubella virus), Pestivirus (mucosal disease virus, swine cholera virus, border disease virus), and Bunyavirus genus (Bunyanvera and related viruses, California encephalitis group virus) Butterfly virus, Rift Valley fever virus), Nairovirus genus (Crimea-Congo hemorrhagic fever virus, Nairobi sheep disease virus), and Ukuvirus genus (Uulcuun)
the Bunyaviridae family, including iemi) and related viruses), and Inf
genza virus genus (influenza virus type A, many human subtypes),
Orthomyxoviridae family, including swine influenza virus, avian influenza virus, and equine influenza virus, influenza B (many human subtypes), and influenza C (probably another genus);
Paramyxovirus genus (parainfluenza virus type 1, Sendai virus, erythrocyte adsorbed virus, parainfluenza virus type 2 to 5, Newcastle disease virus, mumps virus), Morbilillivirus genus (measles virus, subacute sclerosing panencephalitis virus) , Distemper virus, rinderpest virus), Pneum
paramyxoviridae family, including the genus Ovirus (respiratory syncytial virus (RSV), bovine respiratory syncytial virus, and pneumonia virus), and Vesicu
genus Lovirus (VSV) (Chandipura virus, Flanders-Hart Park virus), Ly
Rh including the genus sasavirus (rabies virus), fish Rhabdovirus, and two possible Rhabdoviruses (Marburg virus and Ebola virus)
the Adenaviridae family, including the abdoviridae family, the lymphocytic choriomeningitis virus (LCM), the Tacaribe virus complex, and the Lassa fever virus, and the infectious bronchitis virus (IBV), hepatitis virus, human intestinal coronavirus,
And Coronoaviridae including feline infectious peritonitis (feline coronavirus)
And family. Illustrative DNA viruses that are antigens in vertebrates include:
Although not limited, Orthopoxvirus genus (major pressure ulcer, small pressure ulcer, monkey pressure ulcer vaccinia, cow gourd, buffalo moth, rabbit heel, ectromelia), Leporipoxviru
genus s (myxoma, fibroma), genus Avipoxvirus (fowlpox, other avian pox viruses), genus Capripoxvirus (sheep moth, goat moth), genus Suipoxvirus (pig moth), genus Parapoxvirus (infectious pustular dermatitis virus) , Fake cowpox, bovine papule stomatitis virus) and Iridoviridae
Family (African swine fever virus, frog virus type 2 and type 3, fish lymphocyst virus) and alpha-Ierpesviruses (herpes simplex type 1 and type 2, varicella zoster, equine herpes virus, equine herpes virus type 2) And type 3, pseudorabies virus, bovine infectious keratoconjunctivitis virus, bovine infectious rhinotracheitis virus, feline rhinotracheitis virus, infectious laryngotracheitis virus), Beta-herpesviruses
(Human cytomegalovirus and porcine and monkey cytomegalovirus), ga
Herp containing mma-herpesviruses (Epstein-Barr virus (EBV), Marek's disease virus, squirrel monkey herpes, spider herpes virus, cottontail rabbit herpes virus, guinea pig herpes virus, Ryuke cancer virus)
The esviridae family and the genus Mastadenovirus (human subgroups A, B, C, D, E, and unclassified, simian adenovirus (at least 23 serotypes), infectious canine hepatitis, and cattle, pigs, sheep, frogs And many other species of adenoviruses), the Adenoviridae family, including the genus Avianovirus (avian adenovirus), and non-culturable adenoviruses, and Papillomav
Irus genus (human papilloma virus, bovine papilloma virus, rabbit-shoop papilloma virus, various pathogenic papilloma viruses of other species), Poly
Other primate polyoma viruses such as omavirus (polyomavirus, monkey vacuolating pathogen (SV-40), rabbit vacuolating pathogen (RKV), K virus, BK virus, JC virus, and lymphoproliferative papilloma virus ) Including Papoviri
The dae family and the genus Adeno-associated viruses, Parv
and the Parvoviridae family including the genus Ovirus (feline panleukopenia virus, bovine parvovirus, canine parvovirus, Aleutian mink disease virus, etc.). Finally, DNA viruses may include viruses that are not compatible with the above family, such as Kuru and Creutzfeldt-Jakob disease viruses, and chronic infectious neuropathic factors (CHINA viruses).

Immunostimulatory nucleic acids can also be used to induce immune responses such as antigen-specific immune responses in birds such as hens, chickens, turkeys, ducks, geese, quails, and pheasants. Birds are a major target for many types of infections.

Hatched birds are exposed to pathogenic microorganisms immediately after birth. These birds are initially protected against pathogens by maternally derived antibodies, but this protection is temporary and the bird's own immune system must begin to protect them against the pathogen. It is desirable to protect the infection in young birds at the most susceptible time. When birds live in closed areas and the disease spreads rapidly, it is desirable to prevent infection even in older birds. Accordingly, it is desirable to administer immunostimulatory nucleic acids and non-nucleic acid adjuvants of the present invention to birds to enhance the antigen-specific immune response when antigen is present.

An example of a common infection in chickens is the chicken infectious anemia virus (CIAV).
CIAV was first isolated in Japan in 1979 during the suspension of Marek's disease vaccination investigation (Yuasa et al., 1979, Avian Dis. 23: 366-38).
5). Since that time, CIAV has been detected in commercial poultry in all major countries producing poultry (van Bullow et al., 1991, pp. 690-699).
, Diseases of Poultry, 9th edition, Iowa St
ate University Press).

CIAV infection results in clinical disease characterized by anemia, bleeding, and immunosuppression in young susceptible chickens. Thymic and bone marrow atrophy and consistent lesions of CIAV infected chickens are also characteristic of CIAV infection. Lymphocyte depletion within the thymus and often within the Fabricius sac results in immunosuppression and increases the susceptibility to secondary viral, bacterial, or fungal infections, which subsequently exacerbates the course of the disease. Immunosuppression may cause disease deterioration after infection with one or more of Marek's disease virus (MDV), infectious bursal disease virus, reticuloendotheliosis virus, adenovirus, or reovirus. The pathogenesis of MDV has been reported to be enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proc
edings of the 38th Western Poultry Dise
ases Conference, Tempe, Ariz. ). Furthermore, CIAV has been reported to exacerbate signs of infectious bruza disease (Rosenberger et al.
al. , 1989, Avian Dis. 33: 707-713). Chicken is CA
Develop aged resistance to diseases experimentally induced by A. This is essentially complete by 2 weeks of age, but older birds are still susceptible to infection (Yu
asa, N .; et al. 1979 (supra); Yuasa, N .; et al. ,
Arian Diseases 24, 202-209, 1980). However, when chickens are doubly infected with CAA and immunosuppressants (IBDV, MDV, etc.), aging resistance to the disease is delayed (Yuasa, N. et al., 1979 and 1980 (supra)); von V. et al., J. Veterinary Medical
ine 33, 93-116, 1986). Properties of CIAV that can enhance disease transmission include environmental inactivation and high resistance to several common fungicides. The economic impact of CIAV infection on the poultry industry is evident from the fact that 10% to 30% of infected birds die from disease onset.

As with other vertebrates, bird vaccination can also be performed at any age. Typically, vaccination is done by 12 weeks of age for live microorganisms and 14-18 weeks of age for inactivated microorganisms or other types of vaccines. In ovum
o) For vaccination, vaccination will occur in the last quarter of embryo development. Vaccines can be administered subcutaneously, sprayed, orally, intraocularly, intratracheally, nasally, or by other mucosal delivery methods described herein. Thus, an immunostimulatory nucleic acid of the invention can be administered to birds and other non-human vertebrates using a normal vaccination schedule, and after an appropriate period of time, the antigen as described herein. Can be administered.

Cattle and livestock are also susceptible. The diseases that affect these animals, especially cattle, cause significant economic losses. The method of the present invention can be used to prevent infection in livestock such as cattle, horses, pigs, sheep and goats.

Cattle can be infected with bovine viruses. Bovine viral diarrhea virus (BVDV)
Is a small positive-strand RNA virus with an envelope and is classified into the pestivirus genus, along with swine cholera virus (HOCV) and sheep border disease virus (BDV). Although pestiviruses were previously classified into the Togaviridae family, several studies have suggested reclassification within the Flaviviridae family, along with the flavivirus and hepatitis C virus (HCV) group (Franki,
et al. , 1991).

Based on cell culture analysis, BVDV, a major bovine pathogen, was converted to cytopathic (CP)
A distinction can be made between cell types and non-cytopathic (NCP) cell types. NCP cell types are more extensive, but both cell types are found in cattle. If a pregnant cow is infected with an NCP strain, the cow will be persistently infected and will give rise to a specifically immunotolerant calf that will spread the virus during its lifetime. Persistently infected cattle die due to mucosal disease, after which both biotypes can be isolated from the animal. Clinical signs can include miscarriage, teratogenesis, and respiratory disease, mucosal disease, and mild diarrhea. In addition, thrombocytopenia associated with the outbreak of herd infectious diseases that can lead to animal death has been described, and the strain associated with this disease appears to be more virulent than traditional BVDV.

The equine herpesvirus (EHV) comprises a group of antigenically distinct biological factors that cause a variety of equine infections ranging from asymptomatic to fatal diseases. These include:
Equine herpesvirus type 1 (EHV-1), which is a ubiquitous pathogen in horses, is included. EHV-1 is associated with miscarriages, respiratory diseases, and infectious diseases of central nervous system disorders. A young horse's first infection of the upper respiratory tract results in a febrile illness that lasts 8-10 days. Because an immunologically experienced mare can re-infect through the airways before the disease can be identified, miscarriages usually occur without warning. Neurological syndromes are associated with respiratory disease or miscarriage and affect animals of any sex at any age, leading to ataxia, weakness, and paralysis of the buttocks (Telford, ER et al. al., Virology 189, 304
-316, 1992). Other EHV's include EHV-2, or previously EHV-
Equine cytomegalovirus, EHV-3, equine rash virus, and EHV-4 that were classified as subtype 2 of 1.

  Sheep and goats can be infected by a variety of dangerous microorganisms, including maedi visna.

Primates such as monkeys, apes and macaques can be infected with simian immunodeficiency virus.
Inactivated cell viruses and cell-free whole simian immunodeficiency vaccines have been reported to allow protection in macaques (Stott et al. (1990) Lancet 3
6: 1538-1541; Dessiers et al. PNAS USA (19
89) 86: 6353-6357; Murphey-Corb et al. (1989
(1989) Science 246: 1293-1297; and Carlson.
et al. (1990) AIDS Res. Human Retroviruses
6: 1239-1246). Recombinant HIV gpl20 vaccine has been reported to allow protection in chimpanzees (Berman et al. (1990) N
nature 345: 622-625).

Cats (both pet and wild cats) are susceptible to various microorganisms. For example,
Feline infectious peritonitis is a disease that occurs in both domestic and wild cats (eg, lions, leopards, cheetahs, and jaguars). When it is desired to prevent infection of this and other types of pathogenic organisms in cats, the method of the present invention can be used to vaccinate cats and protect them from infection.

Domestic cats are infected with a number of retroviruses including, but not limited to, feline leukemia virus (FeLV), feline sarcoma virus (FeSV), endogenous Concornavirus (RD-114), feline syncytial virus (FeSFV). sell. Of these, Fe
LV is the most significant pathogen and causes a variety of symptoms including lymphoid and myeloid neoplasms, anemia, immune-mediated disorders, and immune deficiencies similar to human acquired immune deficiency syndrome (AIDS) . In recent years, a specific replication deficient FeL named FeLV-AIDS
V variants were particularly associated with immunosuppressive properties.

The discovery of feline T lymphotropic lentivirus (also referred to as feline immunodeficiency) was first described in Pedersen et al. (1987) Science 235: 790-79.
3. For the characteristics of FIV, see Yamamoto et al. (198
8) Leukemia, December Supplement 2: 2045-2
155; Yamamoto et al. (1988) Am. J. et al. Vet. Res. 4
9: 1246-1258; and Ackley et al. (1990) J. MoI. Viro
l. 64: 5652-5655. For cloning and sequence analysis of FIV, see Olmsted et al. (1989) Proc. Natl. Acad
. Sci. USA 86: 2448-2452 and 86: 4355-4360.

Feline infectious peritonitis (FIP) is a sporadic disease that occurs unpredictably in livestock and wild felines. While FIP is primarily a disease of domestic cats, it has been diagnosed in lions, American lions, leopards, cheetahs, and jaguars. Smaller wild cats affected by FIP include lynx and caracal, sun cat, and manul. In domestic cats, the disease occurs primarily in young animals, but cats of all ages are susceptible to infection. The peak incidence occurs at 6-12 months of age. A decrease in incidence has been shown at ages 5 to 13 and then increases in cats between 14 and 15 years of age.

Viral, bacterial, and parasitic diseases in fish, crustaceans, or other aquatic forms present serious problems in the aquaculture industry. Due to the high density of animals in hatchery tanks or enclosed marine aquaculture areas, infections can eradicate the vast majority of reserves, for example in fish, crustaceans, or other aquatic morphological facilities There is. Once the disease has occurred, prevention of the disease is a better countermeasure than intervention. Fish vaccination is the only prophylaxis that can provide long-term protection through immunity. Nucleic acid based vaccination is described in US Pat. No. 5,780,448 issued to Davis.

The fish immune system has many features similar to the mammalian immune system, such as the presence of B cells, T cells, lymphokines, complement, and immunoglobulins. Fish have a lymphocyte subclass with roles that appear to be similar in many ways to the roles of mammalian B and T cells. By immersion,
Alternatively, the vaccine can be administered orally.

Aquaculture species include, but are not limited to, fish, crustaceans, and other aquatic animals. Fish includes all vertebrate fish and can be teleosts or cartilaginous fish (eg, salmonids, carp, catfish, yellowtail, Thai, and grouper). Salmonids are a family of fish that includes trout (including rainbow trout), salmon, and alpine water swallows. Examples of crustaceans include, but are not limited to clams, lobsters, shrimps, crabs, and oysters. Other aquaculture animals include, but are not limited to, eel, squid, and octopus.

Viral aquaculture pathogen polypeptides include, but are not limited to, viral hemorrhagic sepsis virus (VHSV) glycoprotein (G) or nucleoprotein (N) and infectious hematopoietic necrosis virus (IHNV) G Or N protein, VP1, VP2, VP3, or N structural protein of infectious pancreatic necrosis virus (IPNV), G protein of carp spring viremia (SVC), and membrane association of butina cat virus (CCV) A protein, tegumin or capsid protein or glycoprotein.

Typical parasites that infect horses include Gasterophilus species and Eimeria
leuckarti, Giardia species, and Tritrichomonas equi
Babesia species (RBC), Theileria equi, and Trypanos
Oma species, Klossiella equi and Sarcocystis species.

Typical parasites that infect pigs include Eimeria bebliecki, Ei
meria scabra, Isospora suis, Giardia species and Bal
anticoli, Entamoeba histolytica, and Tox
oplasma gondii and Sarcocystis species, and Trichi
nella spiralis.

Major parasites of dairy and beef cattle include Eimeria species, Cryptosporid
ium sp. Giardia species, Toxoplasma gondii and Bab
esia bovis (RBC), Babesia bigemina (RBC), Tr
ypanosoma species (plasma), Theileria species (RBC), and Theileri
a parva (lymphocytes), Tritrichomonas feet, Sa
and rcocystis species.

Major parasites of raptors include Trichomonas gallinae and Coc
cidia (Eimeria species), Plasmodium relictum, Leu
cocytozoon danilewskyi (owl), Haemoproteu
s species, Trypanosoma species, Histomonas, Cryptospori
dime meleagridis, Cryptospodium baileyi
, Giardia, Eimeria, and Toxoplasma.

Typical parasites that infect sheep and goats include the Eimeria species, Crypt
ospodium sp. Giardia sp. And Toxoplasma g
ondi, Babesia species (RBC), Trypanosoma species (plasma), Th
eilelia species (RBC) and Sarcocystis species.

Typical parasitic infections in poultry include Eimeria acervulina,
E. necatrix, E.M. tenella, Isospora species, and Eimeri
coccidiasis caused by a truncata and Histomonas meleg
histomonas caused by ridis and Histomonas gallinarum, trichomoniasis caused by Trichomonas gallinae, and Hexam
Hexamitosis caused by ita meleagridis. Poultry is Eme
ria maxima, Emeria meleagridis, Eimeria ad
enoeides, Eimeria meleagrimitis, Cryptospo
ridium, Eimeria brunnetti, Emeria adenoeide
s, Leucocytozoon species, Plasmodium species, Hemoproteus
meleagridis, Toxoplasma gondii, and Sarcoc
It can also infect ystis.

The methods of the invention can also be applied to the treatment and / or prevention of parasitic infections in dogs, cats, birds, fish, and ferrets. As a typical avian parasite, Trich
omonas gallinae, Eimeria species, Isospora species, Giar
dia, Cryptospodium, Sarcocystis species, Toxop
lasma gondii, Haemoproteus / Parahaemoprote
us, Plasmodium species, Leucocytozoon / Akiba, Atoxo
plasma and Trypanosoma species. Typical parasites that infect dogs include Trichinella spiralis, Isopola species, Sar
cocystis species, Cryptosporidium species, Hammondia species, Gi
ardia dudenalis (genus canine), Balantidium coli,
Entamoeba histolytica, Hepatozon canis, Toxoplasma gondii, Trypanosoma cruzi, and Ba
besia canis, Leishmania agustigotes, and Neo
and spora caninum.

Typical parasites that infect feline species include the Isospora species, Toxoplasm
a gondii, Sarcocystis sp., Hammondia hammondi
, Besnoitia species, Giardia species, and Entamoeba histolyt
ica, Hepatazoon canis, Cytauxzoon sp. , Cyt
auxzone sp. Cytauxzoon sp. (Erythrocytes, RE cells).

Typical parasites that infect fish include Hexamita, Eimeria, and C
ryptobia species, Nosema species, Myxosoma species, Chidononella
Species, Trichodina Species, Pristophora Species, Myxosoma Hen
neguya, Costaia species, Ichthyophythirius species, Oodin
Ium species.

Typical parasites of wild mammals include Giardia species (carnivores, herbivores), I
sospora species (carnivores), Eimeria species (carnivores, herbivores), and The
Ileria species (herbivores), Babesia species (carnivores, herbivores), Trypan
osoma species (carnivores, herbivores), Schistosoma species (herbivores), F
asciola hepatica (herbivore), Fascioloids magn
a (herbivore), Fasciola gigantica (herbivore), Trichin
and ella spiralis (carnivorous animals, herbivores).

Parasitic infections at the zoo also present serious problems. Bovine family (Bresbok,
Typical parasites of Antelope, Banten, Eland, Gaua, Impala, Kripspringer, Kudu, Gazelle) include Eimeria species. The typical parasites of the subfamily of seals (sea seals, sea lions) include Eimeria phocae
Is mentioned. A typical parasite of the camelid family (camel, llama) is Eim
eria species. Typical parasites of the giraffe family (giraffe) include E
imeria species. Typical parasites of the elephant family (African and Asian elephants) include Fasciola species. Typical parasites of lower primates (chimpanzees, orangutans, apes, baboons, macaques, monkeys) include Giardia sp
. Balantidium coli, Entamoebba histolytic
a, Sarcocystis sp., Toxoplasma gondii, and Plasmo
Dim species (RBC), Babesia species (RBC), Trypanosoma species (plasma)
And Leishmania species (macrophages).

Cancer is one of the leading causes of death in companion animals (ie, cats and dogs).
One. Cancer usually attacks older animals than in domestic pets and is incorporated into the family. Of dogs older than 10 years, 45% are thought to die from the disease. The most common treatment options include surgery, chemotherapy, and radiation therapy. Other treatment modalities used that have achieved some success are laser therapy, cryotherapy, hyperthermia, and immunotherapy. The choice of treatment depends on the type of cancer and the degree of metastasis. Unless the malignant tumor is limited to an isolated area in the body, it is difficult to remove only the malignant tissue without affecting normal cells.

Malignant diseases commonly diagnosed in dogs and cats include, but are not limited to, lymphosarcoma, osteosarcoma, breast tumor, mastocytoma, brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor, Bronchial adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing sarcoma, Wilm tumor, Burkitt lymphoma, microglioma, neuroblastoma, Examples include giant cell tumors of the bone, oral neoplasia, fibrosarcoma, osteosarcoma, and rhabdomyosarcoma. Other neoplasias in dogs include reproductive squamous cell carcinoma, tumors with transmissive darkness, testicular tumors, seminome, sertoli cell tumor, perivascular cell tumor, histiocytoma, melanoma (granulocytic sarcoma) Corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma, basal cell tumor, thymoma, gastric tumor, adrenal carcinoma, oral papillomatosis, hemangioendothelioma, and cystadenoma. Additional malignancies diagnosed in cats include follicular lymphoma, intestinal lymphosarcoma, fibrosarcoma, and lung squamous cell carcinoma. Ferret, a more popular home pet, is known to develop islet cell adenoma, lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALT lymphoma, and gastric adenocarcinoma.

Neoplasias that affect agricultural livestock include leukemia, perivascular neoplasia, and bovine ocular neoplasia (bovine), foreskin fibrosarcoma, ulcerative squamous cell carcinoma, foreskin cancer, connective tissue neoplasia, and mastocytoma (
Horses), liver cancer (pigs), lymphoma and pulmonary adenomatosis (sheep), pulmonary sarcoma, lymphoma,
Rous sarcoma, reticuloendotheliosis, fibrosarcoma, nephroblastoma, B-cell lymphoma, and lymphoid leukemia (avian species) and retinoblastoma, hepatic neoplasia, lymphosarcoma (lymphoblastic lymphoma), Plasmacytoid leukemia, and sarcoma (fish), dairy lymphadenitis (CLA), and chronic, infectious, contact infectious diseases of sheep and goats caused by bacterial sheep pseudotuberculosis, and sheep pulmonary adenopathy And contact infectious lung tumors of sheep.

An allergen refers to a substance (antigen) that can induce an allergic or asthmatic response in a susceptible subject. The list of allergens is enormous and can include pollens, insect venoms, animal dander dust, fungal spores, and drugs (eg, penicillin). Examples of natural animal and plant allergens include, but are not limited to, proteins specific for the following genera: That is, Canine (Canis familyis), Dermato
phagoides (eg, Dermatophagoides farinae),
Felis (Felis domesticus), Ambrosia (Ambrosi)
a artemisfolia), Lolium (eg, Lolium peren)
ne or Lolium multiflorum), Cryptomeria (Cry
ptomeria japonica), Alternaria (Alternaria)
alterna), Alder, Alnus (Alnus glutinoasa)
), Beta (Betula verrucosa), Quercus (Querc)
us alba), Olea (Olea europa), Artemisia (Art
emisia vulgaris), Plantago (eg, Plantago l
anceolata), Parietaria (e.g., Parietaria off)
icinalis or Parietaria judaica), Blatella
(E.g. Blattella germanica), Apis (e.g. Apis
multiflorum), Cuplessus (eg, Cuplessus sem)
pervirens, Cupresus arizonica, and Cupless
us macrocarpa), Juniperus (eg, Juniperus s
abinoides, Juniperus virginiana, Juniperus
communis, and Juniperus ashei), Thuya (e.g.,
Thuya orientalis), Chamaecyclos (eg, Cham
aecaris obtusa), Periplaneta (eg, Peripl)
arena americana), Agropyron (eg, Agropyron).
repens), Secale (e.g., Secreal), Trit
icum (eg, Triticum aestivum), Dactylis (eg, Dactylis glomerata), Festuca (eg, Festuka)
elatior), Poa (eg, Poa privacyis or Poa co
mpressa), Avena (eg, Avena sativa), Holcus (
For example, Holcus lananthus), Anthoxanthum (eg, Ant
hoxanthum odoratum), Arrhenatherum (eg, Ar
rhenatherum elatius), Agrostis (eg, Agrost)
is alba), Phleum (e.g., Phleum platenase), Pha
laris (eg, Phalaris arundinacea), Paspalum
(E.g., Paspalum notatum), Sorghum (e.g., Sorgh)
um halepensis), and Bromus (eg Bromus inner)
mis).

The antigen may be a nucleic acid encoded by a nucleic acid vector or may not be encoded in a nucleic acid vector. In the former case, the nucleic acid vector is administered to the subject, and the antigen is administered in vivo (in vivo).
vo). In the latter case, the antigen can be administered directly to the subject. As used herein, an antigen that is not encoded in a nucleic acid vector refers to any type of antigen that is not a nucleic acid. For example, in some aspects of the invention, the antigen that is not encoded in the nucleic acid vector is a polypeptide. A minor modification of the primary amino acid sequence of a polypeptide antigen can also result in a polypeptide having a substantially equivalent antigenic activity as compared to an unmodified equivalent polypeptide. Such modifications can be deliberate as site-directed mutagenesis or can be spontaneous. All of the polypeptides produced by these modifications are encompassed herein as long as antigenicity still exists. The polypeptide can be, for example, a viral polypeptide.

As used herein, the term “substantially purified”
“purified)” refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates, or other substances with which the polypeptide is naturally associated. One skilled in the art can purify viral or bacterial polypeptides using standard techniques for protein purification. A substantially pure polypeptide will often produce a single major band on a non-reducing polyacrylamide gel. For partially glycosylated polypeptides or those with multiple initiation codons, there may be multiple bands on the non-reducing polyacrylamide gel, but these form a pattern that is unique to polypeptides. The purity of the viral or bacterial polypeptide can also be determined by amino-terminal amino acid sequence analysis. Other types of antigens not encoded by nucleic acid vectors such as polysaccharides, small molecules, mimetics, etc. are described above and are encompassed within the present invention.

The present invention also uses a polynucleotide encoding an antigen polypeptide. It is envisioned that an antigen can be delivered to a subject within a nucleic acid molecule that encodes the antigen such that the antigen is expressed in vivo. Such an antigen that is delivered to a subject in a nucleic acid vector is referred to as an antigen encoded by the nucleic acid vector. A nucleic acid encoding an antigen is operably linked to a gene expression sequence that guides the expression of the antigen nucleic acid in a eukaryotic cell. A gene expression sequence is any regulatory nucleotide sequence, such as a promoter sequence or promoter enhancer combination, that facilitates efficient transcription and translation of an antigen nucleic acid to which the gene expression sequence is operably linked. The gene expression sequence can be, for example, a mammalian or viral promoter such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, promoters for the following genes. Hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, b-actin promoter, and other constitutive promoters. Examples of viral promoters that function constitutively in eukaryotic cells include, for example, cytomegalovirus (
CMV), monkey virus (eg, SV40), papilloma virus, adenovirus,
Examples include human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, Moloney leukemia virus and other retrovirus long terminal repeats (LTR), and promoters from the herpes simplex virus thymidine kinase promoter. Other constitutive promoters are known to those skilled in the art. Promoters useful as gene expression sequences of the present invention include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those skilled in the art.

In general, gene expression sequences can be selected from TATA boxes, capping sequences, C
5 'untranscribed and 5' untranslated sequences involved in transcription and translation initiation, such as AAT sequences, respectively. In particular, such 5 ′ non-transcribed sequences include a promoter region that includes a promoter sequence for transcriptional control of the operably linked antigen nucleic acid. The gene expression sequence is optional,
Include enhancer sequences or upstream activator sequences as desired.

The antigen nucleic acid is operably linked to the gene expression sequence. As used herein, when an antigen nucleic acid sequence and a gene expression sequence are covalently linked so that expression or transcription and / or translation of the antigen coding sequence is under the influence or control of the gene expression sequence, the antigen nucleic acid Sequences and gene expression sequences are said to be operably linked. When transcription of the antigen sequence is caused by the promoter in the 5 ′ gene expression sequence, and (1) the induction of frameshift mutation does not occur due to the nature of the binding between the two DNA sequences, or (2) the antigen sequence of the promoter region Two DNA sequences are said to be operably linked if they do not interfere with the ability to direct the transcription of (3) or interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, if the gene expression sequence can transcribe the antigen nucleic acid sequence so that the resulting transcript is translated into the desired protein or polypeptide, the gene expression sequence is operably linked to the antigen nucleic acid sequence. To do.

The antigen nucleic acid of the invention can be delivered to the immune system alone or in combination with a vector. In a broad sense, a vector is any carrier that can facilitate delivery of an antigen nucleic acid to cells of the immune system so that the antigen can be expressed and presented on the surface of the immune cell. Vectors generally transport nucleic acids into immune cells that have reduced degradation relative to the extent of degradation that results in the absence of the vector. The vector optionally includes the gene expression sequence described above to enhance the expression of the antigen nucleic acid in immune cells. In general, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, or other carriers derived from viral or bacterial sources that have been engineered by insertion or incorporation of antigen nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to, nucleic acid sequences from the following viruses: That is, retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus, adenovirus, adeno-associated virus, SV40 type virus, polyoma virus, Epstein-Barr virus, and papilloma These are viruses, herpes viruses, vaccinia viruses, polioviruses, and RNA viruses such as retroviruses. Although not described, other vectors known in the art can be readily used.

A preferred viral vector is a non-cytopathic eukaryotic virus, wherein the essential gene is replaced with the gene of interest. Non-cytopathic viruses include retroviruses, whose life cycle involves reverse transcription of genomic viral RNA into DNA, followed by proviral incorporation into host cellular DNA. Retroviruses have been approved for human gene therapy trials. The most useful are replication deficient (ie, capable of directing synthesis of the desired protein but not production of infectious particles) retrovirus. Such genetically modified retroviral expression vectors are versatile for high efficiency transduction of genes in vivo. Standard protocol for producing replication-defective retroviruses, including the steps of integrating exogenous genetic material into a plasmid, transfecting a packaging cell line with the plasmid, and producing a recombinant retrovirus with the packaging cell line And the step of recovering virus particles from the tissue culture medium and infecting the target cells with virus particles)
Kriegler, M .; , Gene Transfer and Expression
, A Laboratory Manual W. H. Freeman C.I. O. , Ne
w York (1990) and Molecular Biology, vol. 7, H
umana Press, Inc. , Clifflon, New Jersey (19
91) in Murry, E .; J. et al. Provided with Method.

A preferred virus for certain applications is adeno-associated virus, which is a double-stranded DNA virus. An adeno-associated virus can be engineered into a replication-deficient virus, which can infect a wide variety of cell types and species. In addition, the adeno-associated virus allows heat and lipid solvent stability, high transduction frequency in cells of various lineages including hematopoietic cells, and multiple consecutive transductions because it does not inhibit superinfection Has the advantage of being Reportedly, adeno-associated virus can be integrated into human cellular DNA in a site-specific manner, minimizing the possibility of insertional mutation and the variability of inserted gene expression characteristic of retroviral infection. To. Furthermore, wild-type adeno-associated virus infection continues for more than 100 passages in tissue culture in the absence of selective pressure, suggesting that the adeno-associated virus genome integration is a relatively stable event. Adeno-associated virus can also function in an extrachromosomal manner.

Examples of other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those skilled in the art. For example, Samb
look et al., Molecular Cloning: A Laboratory Ma
nual, Second Edition, Cold Spring Harbor L
See laboratory Press, 1989. In recent years, plasmid vectors have been particularly advantageous for delivering genes to cells in vivo because of their ability to replicate in and integrate into the host genome. There was found. However, these plasmids with promoters that are compatible with the host cell
Peptides can be expressed from genes that are operably encoded in the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pR
C / CMV, SV40, and pBlueScript. Other plasmids are well known to those skilled in the art. In addition, plasmids can be custom designed by removing and adding specific fragments of DNA using restriction enzymes and ligation reactions.

It has recently been discovered that bacteria can be used to deliver a plasmid carrying a gene to the immune system. Modified forms of bacteria, such as Salmonella, can be transfected with plasmids and used as delivery vehicles. Bacterial delivery carriers can be administered to the host subject orally or by other means of administration. Bacteria deliver plasmids to the immune system (eg, B cells, dendritic cells), perhaps by passing through the intestinal wall. Using this methodology, a high level of immune protection was established. Such delivery methods are useful for embodiments of the invention that use systemic delivery of antigens, immunostimulatory nucleic acids, and / or other therapeutic agents.

Thus, in addition to being suitable as a stand-alone drug, immunostimulatory nucleic acids are particularly
Useful as a vaccine adjuvant. It has previously been established that CpG oligonucleotides are good vaccine adjuvants. In order to identify the best immunostimulatory nucleic acids for use as vaccine adjuvants in humans and other non-rodent animals, in vivo screening of different nucleic acids for this purpose was performed. Multiple in vitro (
In vitro assays were evaluated in mice for their predictive value of adjuvant activity in vivo. During the course of this study, in vivo
An in vitro test was identified that would predict efficacy at. On the contrary, it has been found that the activation of both B cells and NK cells is particularly correlated with the ability of immunostimulatory nucleic acids to enhance the in vivo immune response to antigen.

The nucleic acids are also useful for improving dendritic cell survival, differentiation, activation, and maturation. Immunostimulatory nucleic acids have the unique ability to promote cell survival, differentiation, activation, and maturation of dendritic cells. Dendritic progenitor cells isolated from blood by immunomagnetic cell sorting
During the 2-day incubation with GM-CSF, the morphological and functional properties of dendritic cells are developed. Without GM-CSF, these cells undergo apoptosis. Immunostimulatory nucleic acids are superior to GM-CSF in promoting dendritic cell survival and differentiation (MH
CII expression, cell size, particle size). Immunostimulatory nucleic acids also induce dendritic cell maturation.
Tree cells form a link between the innate and acquired immune systems by presenting antigens and through the expression of pattern recognition receptors that detect bacterial molecules such as LPS in their local environment As such, the ability to activate dendritic cells with immunostimulatory nucleic acids has been shown to be in vivo and ex vivo (ex-v) against disorders such as cancer and allergic diseases or infections.
ivo) Support the use of these immunostimulatory nucleic acid based strategies for immunotherapy.
Immunostimulatory nucleic acids are also useful for activating and inducing dendritic cell maturation.

Immunostimulatory nucleic acids also have natural killer cytolytic activity and antibody-dependent cytotoxicity (A
DCC). ADCC can be performed using immunostimulatory nucleic acids in combination with antibodies specific for cellular targets such as cancer cells. When an immunostimulatory nucleic acid is administered to a subject with an antibody, the subject's immune system is induced to kill tumor cells. Antibodies useful in this ADCC procedure include antibodies that interact with cells in the body. Many such antibodies specific for cellular targets have been described in the art and many are commercially available. Examples of these antibodies are listed below in the list of cancer immunotherapy.

The nucleic acids are also useful for reinvoking an immune response from a Th2 immune response to a Th1 immune response. Re-eliciting an immune response from a Th2 immune response to a Th1 immune response can be assessed by measuring the level of cytokines generated in response to this nucleic acid (eg, inducing monocyte cells and other cells) IL-12, IFN-γ and GM-C
Producing Th1 cytokines containing SF). Re-elicitation or re-equilibration of the immune response from a Th2 to Th1 response is particularly useful for the treatment or prevention of asthma. For example, an effective amount for treating asthma can be that amount effective to re-initiate a Th2-type immune response associated with asthma into a Th1-type response. Th2 cytokines, especially IL-4 and I
L-5 is elevated in the airways of asthmatic subjects. These cytokines promote important aspects of the asthmatic inflammatory response, including IgE isotype switching, eosinophil chemotaxis and activation, and mast cell growth. Th1 cytokines, especially IFN-γ and IL-12,
Th2 clone formation and Th2 cytokine production can be suppressed. The immunostimulatory nucleic acids of the present invention cause an increase in Th1 cytokines that help re-equilibrate the immune system, primarily preventing or reducing the side effects associated with Th2 immune responses.

The present invention also includes methods for using immunostimulatory nucleic acids to induce broad spectrum resistance to antigen non-specific and innate immune activation and infectious challenges. As used herein, the term antigen non-specific and innate immune activation refers to the activation of immune cells other than B cells, and for example, NK cells, T cells or others that can respond in an antigen independent manner Activation of any immune cell or some combination of these cells. Broad spectrum resistance to infectious challenges is induced because immune cells are in an active form and primed to respond to any invading compound or microorganism. These cells need not be specifically primed for a particular antigen. This is particularly useful in bacterial warfare and other situations described above, such as travelers.

The nucleic acid of the present invention includes antibacterial agents, adjuvants, cytokines, anticancer treatments, allergy medicines,
It can be used in combination with other therapeutic agents including asthma medicine.

The nucleic acids of the invention can be administered to a subject along with an antimicrobial agent. As used herein,
Antibacterial agents refer to naturally occurring or synthetic compounds that can kill or inhibit infectious microorganisms. The type of antibacterial agent useful according to the invention depends on the type of microorganism that the subject is infected or is at risk of becoming infected. Antibacterial agents include, but are not limited to, antibacterial agents, antiviral agents, antifungal agents and antiparasitic agents. "Anti-infectives"
Phrases such as "antibacterial agent", "antiviral agent", "antifungal agent", "antiparasitic agent" and "parasiticidal agent" have well-established meanings for those skilled in the art, It is defined in standard medical textbooks. In summary, antibacterial drugs kill or inhibit bacteria and include antibiotics and other synthetic or natural compounds with similar functions.

Antibiotics are low molecular weight molecules that are produced as secondary metabolites by cells such as microorganisms. In general, antibiotics interfere with the function or structure of one or more bacteria that are specific for the microorganism and are not present in the host cell. Antiviral agents are isolated from natural sources,
Or can be synthesized and is useful for killing or inhibiting viruses. Antifungal agents are used to treat superficial fungal infections and opportunistic and primary systemic fungal infections. Antiparasitic agents kill or inhibit parasites.

Antibacterial agents inhibit bacterial killing or bacterial growth or function. A large class of antibacterial agents are antibiotics. Antibiotics that are effective in killing or inhibiting a wide range of bacteria are termed broad spectrum antibiotics. Other types of antibiotics are preferentially effective against Gram positive or Gram negative classes of bacteria. These types of antibiotics are
It is called a narrow spectrum antibiotic.

Other antibiotics that are effective against a single microorganism or disease and not effective against other types of bacteria are termed limited spectrum antibiotics. Antibacterial agents are often classified based on their primary mode of action. In general, antibacterial agents are cell wall synthesis inhibitors, cell membrane inhibitors, protein synthesis inhibitors, nucleic acid synthesis inhibitors or functional inhibitors, and competitive inhibitors.

Antibacterial agents useful in the present invention include, but are not limited to: Natural penicillin, semi-synthetic penicillin, clavulanic acid, cephalosporin, bacitracin, ampicillin, carbenicillin, oxacillin, azulocillin, mezlocillin, piperacillin, methicillin, dicloxacillin, nafcillin, cephalothin, cephapillin, cephaloxin, cephalazol, cephalordol Cefuroxin, cefoxitin, cefotaxime,
Cefrosin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidin, moxalactam, carbapenem, imipenem, monobactem, euthreonam, vancomycin, polymyxin, amphotericin B, nystatin, imidazole, clotrimazole, miconazole, ketoconazole, miconazole, ketoconazole , Tetracycline, chloramphenicol, macrolide, aminoglycoside, streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline, minocycline, doxycycline, chlorotetracycline, erythromycin, roxithromycin, clarithromycin, oleandromycin, azis Mycin, chloramphenicol, quinolone, cotrimoxazole, norfloxacin, ciprofloxacin, enoxacin, nalidixic acid, temafloxacin, sulfonamide, gantricin, and trimethoprim; acedapson; sodium acetosulfone; alamesin; alexidine; Amycline; amifoxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; sodium aminosalicylate; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apramycin; Astromycin sulfate; Avila Machine; Avoparcin; Azithromycin; Azulocillin sodium; bacamicillin hydrochloride; bacitracin; bacitracin methylene disalicylate; bacitracin zinc; bumbermycin; benzoylpath calcium; berylomycin; bentamicin sulfate; biapenem; vinylamycin; bifenamide hydrochloride; Butyrosine sulfate; capreomycin sulfate; carbadox; carbenicillin disodium; carbenicillin indanyl sodium; carbenicillin phenyl sodium; carbenicillin potassium; carmonam sodium; cefaclor; cefadorxil; cefamandole; Dole sodium; cephaparol; cephatrizine; Cefazolin sodium; cefbuperazone; cefdinir;
Cefepime; cefepime hydrochloride; cephetecol; cefixime; cemenoxime hydrochloride;
Cefmetazole; cefmetazole hydrochloride; cefoniside monosodium; cefoniside sodium; cefoperazone sodium; cefolanide; cefotaxime sodium; cefotetan; cefotetan disodium; cefothiam hydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; Cefpyramid sodium; cefpyrom sulfate; cefpodoxime proxetyl; cefprodil;
Cefloxazine; cefsulodin sodium; ceftazidime; ceftibbutene; ceftizoxime sodium; ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cefacetril sodium; cephalexin; cephalexin hydrochloride; Glycine; cephaloridine; cephalotin sodium; cefapirin sodium; cephalazine; cetocycline hydrochloride; cetophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium Succinate; chlorhexidine phosphanylate; chloroxylenol; chlortetracycline bisulfate; chlortetracycline Ciprofloxacin; ciprofloxacin hydrochloride; silolemycin; clarithromycin; clinafloxacin hydrochloride; clidamycin; clidamycin hydrochloride; clindamycin palmitate hydrochloride; clindamycin phosphate; clofazimine; Cloxicillin sodium; colistimate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine;
Demeclocycline; Demeclocycline hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin sodium; Dihydrostreptomycin sulfate; Dipyrithion; Sodium; Enoxacin; Epicillin; Epitetracycline HCl; Erythromycin; Erythromycin Assist Rate; Erythromycin Estrate; Erythromycin Inethyl Succinate; Erythromycin Single Sceptate; Hydrochloric acid; Ethionamide; Fleloxacin; Floxacillin; Fludaran; Flumequine; Fosfomycin; Phosfomycin Tromethamine; Fumocillin; Furazolium chloride; Furazolium tartrate; Fusidate sodium: Fusidic acid; Gentamicin sulfate; Gramicidin; Haloprogine; Hetacillin; Hetacillin potassium; Hexedine; Ivafloxacin; Imipenem; Isoconazole; Isepamicin;
Loxithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; lomefloxacin mesylate; loracarbef; mafenide; meclocycline; Metacycline hydrochloride; methenamine; methenamine hippuric acid; methenamine mandelic acid; methicillin sodium; metioprim; metomenazole hydrochloride; metronidazole phosphate; Sodium phosphate; sodium nalidixate; nalidixic acid; natamycin; nebramycin; Neomycin sulfate; neomycin undecylate; netilmicin sulfate; neuramycin; niphraden; nifuralzone; Norfloxacin; novobiocin sodium; ofloxacin; ormethoprim; oxacillin sodium; oximonam; oximonam sodium; oxophosphate; oxytetracycline calcium; oxytetracycline calcium; oxytetracycline hydrochloride; ; Penicillin G benza Emissions; Penicillin G Potassium;
Penicillin G procaine; penicillin G sodium; penicillin V; penicillin V benzathine; penicillin V hydrabamine; penicillin V potassium; pentididin sodium; phenylaminosalicylic acid; piperacillin sodium; pirbenicillin sodium; Pibampicillin mopate; pivampicillin probe; polymyxin B sulfate; porphyromycin;
Propicacin; pyrazine amide; pyrithione zinc; quindecamine acetate; quinupristin;
Raphephenicol; ramoplanin; ranimycin; relomycin; repromicin; rifabutin; rifamethane; rifamexil; rifamidine; rifapentine; rifaximin; rifaximin; loritetracycline;
Rosalamycin butyric acid; Rosalamycin propionic acid; Rosalamycin sodium phosphate; Rosalamycin stearic acid; Rosoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfetrine sodium; Salmoxycillin; Salpicillin;
Sisomycin; Sisomycin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;
Spiramycin; stalinomycin hydrochloride; stepinomycin; streptomycin sulfate; streptnicozide; sulfabenz; sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfatisine; Sulfamethazine; sulfamethoxazole; sulfamonomethoxoxine; sulfamoxol; sulfanilate zinc; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; Sulfisoxazole; sulfisoxazole acetyl; sulfisoxazolediolamine; sulfomyxin; Sultamicillin; sancillin sodium; talampicin hydrochloride; teicoplanin; temafloxacin hydrochloride; temosillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprim; tiamphenicol; tifecillin potassium; Ticarcillin monosodium; ticlaton; thiodonium chloride; tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidine; troleandomycin sulfate; tyrosricin; bancomacin; Mycin; and zolvamycin.

Antiviral agents are compounds that prevent infection of cells by viruses or replication of viruses within cells. There are many antiviral drugs but fewer than antibacterial drugs. The process of viral replication is closely related to DNA replication in the host cell, since non-specific antiviral agents are often toxic to the host. There are several processes within the process of viral infection that can be blocked or inhibited by antiviral agents. These stages include viral attachment to host cells (immunoglobulin or binding peptide), viral uncoating (
Including, for example, amantadine), viral mRNA synthesis or translation (eg, interferon), viral RNA or DNA replication (eg, nucleoside analogs), maturation of novel viral proteins (eg, protease inhibitors), and viral budding and release. .

Nucleotide analogs are synthetic compounds that are similar to nucleotides, but have incomplete or unusual deoxyribose or ribose groups. Once the nucleotide analogs are in the cell, they are phosphorylated to produce the formed triphosphate that competes with normal nucleotides for incorporation into viral DNA or RNA. Once the triphosphate form of the nucleotide analog is incorporated into the elongating nucleic acid strand, it irreversibly associates with the viral polymerase and hence causes chain termination. Nucleotide analogs include, but are not limited to, acyclovir (used for the treatment of herpes simplex virus and varicella-zoster virus), ganciclovir (useful for the treatment of cytomegalovirus), idoxuridine, ribavirin (treatment of RS virus) Useful), dideoxyinosine, dideoxycytidine, and zidovudine (azidothymidine). Interferons are cytokines that are secreted by virus-infected cells and immune cells. Interferons function by binding to specific receptors on cells adjacent to the infected cell, causing changes in the cell that protect it from viral infection. α-interferon and β-interferon also induce the expression of class I and class II MHC molecules on the surface of infected cells, causing increased antigen presentation for immune cell recognition. α-interferon and β-interferon are available in recombinant form and have been used for the treatment of chronic hepatitis B and C infection. At dosages that are effective for antiviral therapy, interferon has serious side effects such as fever, malaise and weight loss.

Immunoglobulin therapy has been used to prevent viral infections. Immunoglobulin therapy for viral infection is different from bacterial infection. Because, rather than being antigen-specific, this immunoglobulin therapy works by binding to extracellular virions and preventing them from attaching and invading cells susceptible to viral infection. This treatment is useful to prevent viral infection for the period of time in which antibodies are present in the host. In general, there are two types of immunoglobulin therapy, normal immunoglobulin therapy and hyperimmunoglobulin therapy. Normal immunoglobulin therapy utilizes antibody products prepared and pooled from serum of normal blood donors. This pooled product contains low titers of antibodies against a broad range of human viruses such as hepatitis A, parvovirus, enterovirus (especially in neonates). Hyperimmunoglobulin therapy utilizes antibodies prepared from the sera of individuals with high titers of antibodies against specific viruses. These antibodies are
It is then used against a specific virus. Examples of hyperimmune globulin are herpes zoster immunoglobulin (useful for prevention of chickenpox in tolerant infants and newborns), human rabies immunoglobulin (useful for prevention after exposure to subjects bitten by violent animals), hepatitis B Includes immunoglobulins (useful in the prevention of hepatitis B virus, particularly in subjects exposed to the virus), and RSV immunoglobulins (useful in the treatment of RS virus infection).

Another type of immunoglobulin therapy is active immunization. This includes administration of antibodies or antibody fragments to viral surface proteins. Two types of vaccines available for active immunization of hepatitis B include serum-derived hepatitis B antibodies and recombinant hepatitis B antibodies. Both are prepared from HBsAg. These antibodies are administered in three doses to subjects at high risk of infection with hepatitis B virus, such as healthcare workers, chronic carrier sexual partners, and infants.

Thus, high viral agents useful in the present invention include, but are not limited to, immunoglobulins, amantadine, interferons, nucleoside analogs, and protease inhibitors. Detailed examples of antiviral agents include, but are not limited to: acemannan; acyclovir;
Acyclovir sodium; adefovir; alovudine; alvirceptos dotox; anantadine hydrochloride; alanothine; Famciclovir; Famotine hydrochloride; Fiacitabine; Fiarridine; Fosalylate; Foscarnet sodium; Phosfonet sodium; Ganciclovir; Ganciclovir sodium; Idoxuridine; Ketoxal; Pirodavir; ribavirin; rimantadine hydrochloride; saquinavirme Rate; Somantajin hydrochloride; Soribuujin; Sutatoron;
Stavudine; Tyrolone hydrochloride; Trifluridine; Valaciclovir hydrochloride; Vidarabine; Vidarabine phosphate; Vidarabine sodium phosphate; Biloxam; Zalcitabine; Zidovudine;
And gimbioxime.

Antifungal agents are useful for the treatment and prevention of infectious fungi. Antifungal agents are sometimes classified according to their mechanism of action. Some antifungal agents function as cell wall inhibitors by inhibiting glucose synthesis. These include (but are not limited to) Basiungin / ECB. Other antifungal agents function by destabilizing membrane integrity. These include imidazole, such as clotrimazole, sertaconazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole, and FK463, amphotericin B, BAY 38-95.
02, MK991, pradimicin, UK292, butenafine, and terbinafine (but not limited to). Other antifungal agents function by breaking down chitin (eg, chitinase) or by immunosuppression (501 cream). Some examples of commercially available drugs are shown in Table 3.

Accordingly, antifungal agents useful in the present invention include imidazole, FK463, amphotericin B, BAY 38-9502, MK991, pradimicin, UK292, butenafine, chitinase, 501 cream, acrisolcin; ambletisine; amorolfine, amphotericin B; azaconazole; Bifungine; bifonazole; bifenamine hydrochloride; bispyrithione magsulfex; butconazole nitrate; calcium undecylenate; candicidin; fuchsin carboxylic acid; chlordantoin; cyclopirox; cyclopyrox olamine; sirofungin; cisconazole; clotrimazole;
Cuprimixin; denofungin; dipyrithione; deconazole; econazole; econazole nitrate; enilconazole; etonam nitrate; fenticonazole nitrate; Mepartricin; miconazole; miconazole nitrate; monensin; monensin sodium; naphthifine hydrochloride; neomycin undecylenate;
Nitralamine hydrochloride; Nistatin; Octanoic acid; Orconazole nitrate; Oxyconazole nitrate; Oxyfungin hydrochloride; Parconazole hydrochloride; Partricin; Potassium iodide; Prochronol; Sinefungin; sulconazole nitrate; terbinafine; terconazole; thiram; ticlaton; thioconazole;
Tolnadate; tolnaftate; triacetin; triafungin; undecylenic acid; biridflugin; zinc undecylenate; and dinoconazole hydrochloride.

Examples of anti-parasitic agents, also called parasiticides useful for human administration, include albendazole, amphotericin B, benznidazole, bitionol, chloroquine hydrochloride, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarba Mazine, diloxanide furoate, eflornithine, furazolidaone, glucocorticoid, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine,
Meglumine antimonate, melarsoprole, metriphonate, metronidazole, niclosamide, niflutimox, oxamuniquin, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyranterpamoate, pyrimethamine sulfonamide, Pyrimethamine sulfadoxine, quinacrine hydrochloride, quinine sulfate, quinidine gluconate, spiramycin, sodium stibogluconate (sodium antimony gluconate), suramin, tetracycline, doxycycline, thiabendazole, tinidazole, trimethoprim
m) -sulfamethoxazole, and tripalsamide, some of which are used alone or in combination with others, but are not limited to.

Parasiticides used in non-human subjects include piperazine, diethylcarbamadin, thiabendazole, fenbendazole, albendazole, oxyfendazole, oxybendazole, fevantel, levamisole, pyrantel tartrate, pyrantel pamoate , Dichlorvos, ivermectin, dorametic, milbemycin oxime,
Iprinomectin, moxidectin, N-butyl chloride, toluene, sodium hygromycin B thiacetalsemide, melarsomin, praziquantel, epsiprantel, benzimidazole (eg, fenbendazole, albendazole, oxyfendazole, chlorthrone, albendazole, amprolium) , Decoquinate, lasaloside, monensin, sulfadimethoxine, sulfamethazine, sulfaquinoxaline, and metronidazole.

Parasiticides used in horses include mebendazole, oxyfendazole, fevantel, pyrantel, dichlorvos, trichlorfone, ivermectin, piperazine; For Westeri: ivermectin, benzimidazole (eg thiabendazole, cambendazole, oxybendazole, and fenbendazole)
. Useful parasiticidal agents for dogs include milbemycin oxime (oxi)
ne), ivermectin, pirantel pamoate, and a combination of ivermectin and pirantel. Treatment of parasites in pigs may include the use of levamisole, piperazine, pirantel, thiabendazole, dichlorvos, and fenbendazole. In sheep and goats, anthelmintic agents include levamisole or ivermectin. Capolsolate is the cat's D.C. It has shown some efficacy in the treatment of immitis.

Immunostimulatory nucleic acids can also be administered in combination with anti-cancer treatments. Anti-cancer therapies include cancer drugs, radiation, and surgical procedures. As used herein, “cancer therapeutic agent” refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, “treatment of cancer” includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of a cancer that has developed. In other aspects, a cancer therapeutic is administered to a subject at risk of developing cancer for the purpose of reducing the risk of developing the cancer. Various medicaments for the treatment of cancer are described herein. For purposes herein, cancer therapeutics are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal treatments, and biological response modifiers.

As used herein, “cancer therapeutic agent” refers to an agent that is administered to a subject for the purpose of treating cancer. As used herein, “treatment of cancer” includes preventing the development of cancer, reducing the symptoms of cancer, and / or inhibiting the growth of a cancer that has developed. In other aspects, a cancer therapeutic is administered to a subject at risk of developing cancer for the purpose of reducing the risk of developing the cancer. Various medicaments for the treatment of cancer are described herein. For purposes herein, cancer therapeutics are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormonal treatments, and biological response modifiers. In addition, the methods of the present invention are intended to include the use of more than one cancer therapeutic with an immunostimulatory nucleic acid. As one example, where appropriate, immunostimulatory nucleic acids can be administered with both chemotherapeutic and immunotherapeutic agents. Alternatively, the cancer therapeutic agent is an immunotherapeutic agent and cancer vaccine, or chemotherapeutic agent and cancer vaccine, all administered to a single subject for the purpose of treating a subject having cancer or at risk of developing cancer. Or a chemotherapeutic agent, an immunotherapeutic agent, and a cancer vaccine.

Cancer therapeutics can function in a variety of ways. Some cancer therapeutics work by targeting physiological mechanisms specific to tumor cells. Examples include specific genes that are mutated in cancer,
And such genes, which may be targeted to the products of those genes (ie mainly proteins) include oncogenes (eg Ras, Her2, bcl-2), tumor suppressor genes (eg EGF, p53) , Rb), and cell cycle targets (eg, CDK4
P21, telomerase), but is not limited thereto. Cancer therapeutics can alternatively target signaling pathways and molecular mechanisms that are altered in cancer cells. Targeting cancer cells through epitopes expressed on their cell surface is achieved through the use of monoclonal antibodies. This latter type of cancer therapeutic is generally referred to herein as immunotherapy.

Other cancer therapeutics target cells other than cancer cells. For example, some medications stimulate the immune system to attack tumor cells (ie, cancer vaccines). Still other medications, called angiogenesis inhibitors, function by attacking the blood supply of solid tumors. Because most malignant cancers can metastasize (ie, they are present at the primary tumor site and disseminated in distant tissue to form secondary tumors), drugs that interfere with this metastasis are also Useful for treatment. As angiogenesis mediators, basic FGF, VEGF, angiopoietin, angiostatin, endostatin, TNFα, TNP-470, thrombospondin-1
, Platelet factor 4, CAI, and specific members of integrin family proteins. One class of drugs of this type are metalloprotease inhibitors, which inhibit the enzymes used by cancer cells from being present at the primary tumor site and overflowing into another tissue.

An immunotherapeutic agent is a medicament derived from an antibody or antibody fragment that specifically binds to or recognizes a cancer antigen. As used herein, a cancer antigen is broadly defined as an antigen expressed by cancer cells. Preferably, the antigen is expressed on the cell surface of cancer cells. Even more preferably, the antigen is an antigen that is not expressed on normal cells or at least not at the same level as cancer cells. Antibody-based immunotherapy can function by attacking cancer cells by binding to the cell surface of cancer cells and thereby stimulating the underlying immune system. Another way in which antibody-based immunotherapy works is as a delivery system for specific targeting of toxic substances to cancer cells. Antibodies are usually lysine (eg, derived from castor bean), calicheamicin.
And toxins such as maytansinoids, radioisotopes such as iodine 131 and yttrium 90, chemotherapeutic agents (as described herein), or biological response modifiers and conjugates It becomes. In this way, toxic substances can be enriched in the cancer area and non-specific toxicity to normal cells can be minimized. In addition to the use of antibodies specific for cancer antigens, antibodies that bind to the vasculature (eg, antibodies that bind to endothelial cells) are also useful in the present invention. This is because solid tumors generally survive depending on newly formed blood vessels, so most tumors can reinforce and stimulate the growth of new blood vessels. As a result, one strategy of many cancer mechanisms is to attack the blood vessels that feed the tumor and / or attack the connective tissue (or stroma) that supports such blood vessels.

The use of immunostimulatory nucleic acids in combination with immunotherapeutic agents (eg, monoclonal antibodies) has led to many mechanisms (discussed above), including significant enhancement of ADCC, natural killer (
NK) Long-term survival can be prolonged through cell activation and increased IFNα levels. Nucleic acids used in combination with monoclonal antibodies act to reduce the dose of antibody required to achieve biological results.

  Examples of cancer immunotherapy currently in use or in development are listed in Table 4.

Still other types of chemotherapeutic agents that can be used in accordance with the present invention include aminoglutethimide, asparaginase, busulfan, carboplatin, chloroumbucil, cytarabine hydrochloride, dactinomycin, daunorubicin hydrochloride, estramustine phosphate sodium, etoposide (VP16 -213), floxuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxycarbamide), ifosfamide, interferon α-2a, interferon α-2b, leuprolide acetate (LHRH release factor analog), lomustine (CCNU), Mechlorethamine hydrochloride (nitrogen mustard), mercaptopurine, mesna, mitoten (op'-DDD), mitozantrone hydrochloride, octreotide, plicamycin, hydrochloric acid Rokarubajin, streptozocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, Ansakurin (m-AMSA)
, Azacitidine, erythropoietin, hexamethylmelamine (HMM), interleukin 2, mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), pentostatin (2'deoxycoformycin), semustine (methyl-CCNU) ), Teniposide (VM-26), and vindesine sulfate.

A cancer vaccine is a medicament intended to stimulate an endogenous immune response against cancer cells. Currently produced vaccines primarily activate the humoral immune system (ie, the antibody dependent immune response). Other vaccines currently under development focus on the activation of the cell-mediated immune system, including cytotoxic T cells that can kill tumor cells. Cancer vaccines generally enhance the presentation of cancer antigens to antigen presenting cells (eg, macrophages and dendritic cells) and / or other immune cells (eg, T cells, B cells, and NK cells).

Cancer vaccines can take one of several forms, as discussed below, but these objectives are by APC and by the final presentation of antigen presentation on the cell surface associated with MHC class I molecules In order to promote the endogenous processing of such antigens, the delivery of cancer antigens and / or cancer-associated antigens to antigen presenting cells (APCs). One form of cancer vaccine is a whole cell vaccine, which is a preparation of cancer cells that is removed from a subject, processed in vitro, and then reintroduced into the subject as whole cells. Tumor cell lysates can also be used as cancer vaccines to elicit an immune response. Another form of cancer vaccine is a peptide vaccine, which activates T cells using a cancer-specific small protein or a cancer-associated small protein. Cancer-associated proteins are proteins that are not expressed exclusively by cancer cells (ie, other normal cells can also express these antigens).
However, the expression of cancer-associated antigens is generally consistently up-regulated by certain types of cancer. Yet another form of cancer vaccine is a dendritic cell vaccine, which includes whole dendritic cells exposed in vivo to a cancer antigen or cancer-associated antigen. Dendritic cell lysates or membrane fractions can also be used as cancer vaccines. Dendritic cell vaccines can directly activate antigen presenting cells. Other cancer vaccines include ganglioside vaccines, heat shock protein vaccines, viral and bacterial vaccines, and nucleic acid vaccines.

The use of immunostimulatory nucleic acids in combination with a cancer vaccine has been improved in addition to activating NK cells and endogenous dendritic cells and increasing IFNα levels.
It provides an antigen-specific humoral immune response and an antigen-specific cell-mediated immune response. This enhancement makes it possible to achieve the same effective effect with a vaccine containing a reduced antigen dose. In some cases, the cancer vaccine can be used with an adjuvant as described above.

Other vaccines take the form of dendritic cells (which are exposed to cancer antigens in vitro,
The antigen is processed and cancer antigens related to MHC molecules can be expressed on the cell surface for effective antigen presentation to other immune system cells).

Immunostimulatory nucleic acids are used in one aspect of the invention in combination with dendritic cell based cancer vaccines. Dendritic cells are professional antigen presenting cells. Dendritic cells link the innate and acquired immune systems through the expression of pattern recognition receptors by presenting antigens and detecting microbial molecules such as LPS in their local environment. Form. Dendritic cells efficiently internalize, process, and present soluble specific antigens to which they are exposed. The process of internalization and antigen presentation is a rapid up-regulation of major histocompatibility complex (MHC) and costimulatory molecule expression, cytokine production, and where they are thought to be involved in T cell activation. Cause migration to certain lymphoid organs.

  Table 5 lists various cancer vaccines currently in use or in development.

As used herein, chemotherapeutic agents include immunotherapeutic agents and all other forms of cancer therapeutics that do not fall within the classification of cancer vaccines. As used herein, a chemotherapeutic agent is
Includes both chemical and biological agents. These agents function to inhibit the cellular activity that cancer cells rely on for continued survival. The class of chemotherapeutic agents includes alkylating agents, alkaloid agents, antimetabolites, hormones or hormone analogs, and various antitumor drugs. Most if not all of these drugs are directly toxic to cancer cells and do not require an immune response. The combination of chemotherapy and immunostimulatory nucleic acid administration increases the maximum tolerated dose of chemotherapy.

The chemotherapeutic agents currently in development or in clinical use are shown in Table 6.

In one embodiment, the methods of the invention use immunostimulatory nucleic acids instead of using IFNα therapy in the treatment of cancer. Currently, some treatment protocols require the use of IFNα. Since IFNα is generated following administration of several immunostimulatory nucleic acids, these nucleic acids can be used to endogenously generate IFNα.

In another embodiment, the asthma / allergy medicament comprises a PDE-4 inhibitor, bronchodilator / β-2 agonist, K + channel opener, VLA-4 antagonist, neurokin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonic acid antagonist, A medicament selected from (but not limited to) a 5 lipoxygenase inhibitor, a thrombosin A2 receptor antagonist, a thromboxane A2 antagonist, a 5-lipox activating protein inhibitor, and a protease inhibitor. In some important embodiments, the asthma / allergy medicament is a bronchodilator / β-2 agonist selected from the group consisting of salmeterol, salbutamol, terbutaline, D2522 / formoterol, fenoterol, and orciprenaline.

In another embodiment, the asthma / allergy medicament is a medicament selected from the group consisting of an antihistamine and a prostaglandin inducer. In one embodiment,
Antihistamines include loratidine, cetirizine, buclidin, ceteridine analogues, fexofenadine, terfenadine, desloratadine, norastemisole, epinastine,
It is selected from the group consisting of ebastine, ebastine, astemizole, levocabastine, azelastine, tranilast, terfenadine, mizolastine, betatustine, CS560, HSR609. In another embodiment, the prostaglandin inducer is S-57.
51.

In yet another embodiment, the asthma / allergy medicament is selected from the group consisting of steroids and immunomodulators. Immunomodulators include anti-inflammatory agents, leukotriene antagonists, IL-4 muteins, soluble IL-4 receptors, immunosuppressants, anti-IL-4 antibodies, IL-
4 antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-IL-9 antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, and IgE down-regulators ( However, it is not limited to these). In one embodiment, the IgE down-regulator is anti-IgE.

In another embodiment, the steroid is selected from the group consisting of beclomethasone, fluticasone, tranquinolone, budesonide, and budesonide. In still further embodiments, the immunosuppressive agent is a tolerated peptide vaccine.

In one embodiment, the immunostimulatory nucleic acid is administered at the same time as the asthma / allergy medication. In another embodiment, the subject is a subject who is immunocompromised.

The immunostimulatory nucleic acid is further combined with other therapeutic agents (eg, adjuvants)
It can enhance the immune response. Immunostimulatory nucleic acids and other therapeutic agents can be administered simultaneously or sequentially. When other therapeutic agents are administered at the same time, they can be administered as the same or separate formulations but are administered at the same time. If administration of the other therapeutic agent and the immunostimulatory nucleic acid is separated in time, the other therapeutic agents are administered sequentially with each other and with the immunostimulatory nucleic acid. The separation of time between administrations of these compounds can be in minutes or longer. Other therapeutic agents include adjuvants, cytokines, antibodies,
Antigens, etc. (but not limited to).

The composition of the present invention may also contain a non-nucleic acid adjuvant. A non-nucleic acid adjuvant is any molecule or compound that can stimulate a humoral and / or cellular immune response other than the immunostimulatory nucleic acids described herein. Non-nucleic acid adjuvants include, for example, adjuvants that cause a retention effect, immunostimulatory adjuvants, and adjuvants that cause a retention effect and stimulate the immune system.

As used herein, an adjuvant that causes a retention effect is an adjuvant that slowly releases the antigen into the body, thereby prolonging the exposure of immune cells to the antigen. Adjuvants of this class include alum (eg, aluminum hydroxide, aluminum phosphate); or emulsion based formulations (mineral oil, non-mineral oil, water-in-oil or oil-in-water emulsion, Montanide Adjuvant Se
ppic ISA series (eg Montanide ISA720, AirLiq
oil-in-water emulsions such as uide, Paris, France)); MF-
59 (Squalene-in-water emulsion stabilized with Span 85 and Tween 80; Ch
iron Corporation, Emeryville, CA); and PROVA
X (oil-in-water emulsion with stabilizing surfactant and micelle forming agent; IDEC, Pharma
(but not limited to, the Nationals Corporation, San Diego, CA).

An immunostimulatory adjuvant is an adjuvant that causes activation of cells of the immune system.
This, for example, causes immune cells to produce and secrete cytokines. This class of adjuvants includes Q.I. Saponins purified from the bark of saponaria trees (for example,
QS21 (glycolipid eluting in the 21st peak in the HPLC fraction; Aqua Bio
pharmaceuticals, Inc. , Worcester, MA)); poly [di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Res
arch Institute, USA); derivatives of lipopolysaccharides (eg, monophosphoryl lipid A (MPL; Ribi ImmunoChem Research)
Inc. , Hamilton, MT), muramyl dipeptide (MDP; Ribi), and threonyl muramyl dipeptide (t-MDP; Ribi)); OM-174 (lipid A
Glucosamine disaccharides related to OM; Pharma Pharma SA, Meyrin, S
Witzerland); and Leishmania elongation factor (purified Leishmani)
a protein; Corixa Corporation, Seattle, WA) (but not limited to).

Adjuvants that cause a reservoir effect and stimulate the immune system are compounds that have both of the functions identified above. This type of adjuvant includes ISCOMS (an immunostimulatory complex that contains mixed saponins, lipids, and pore-sized particles that can hold antigen; CSL, Melbourne, Australia); SB
AS2 (SmithKline Beecham Adjuvant System # 2 (oil-in-water emulsion containing MPL and QS21): SmithKline Beecham B
iologicals [SBB], Rixensart, Belgium); SB-AS
4 (SmithKline Beechem adjuvant system # 4 (including alum and MPL): SBB, Belgium); micelle-forming nonionic block copolymer (eg CRL1005), which is hydrophobic sandwiched between polyoxyethylene chains Including a straight chain of polyoxypropylene; Vaxcel, Inc. , Norcross, G
A); and Syntex Adjuvant Formulation (SAF, T
an oil-in-water emulsion comprising ween 80 and a nonionic block copolymer; Syntex
Chemicals, Inc. , Boulder, CO) (but not limited to).

Immunostimulatory nucleic acids are useful as adjuvants to induce a humoral immune response by themselves. Thus, they can be delivered to a subject that has been exposed to an antigen, resulting in an enhanced immune response to the antigen.

Immunostimulatory nucleic acids are useful as mucosal adjuvants. It has already been discovered that both systemic and mucosal immunity are induced by mucosal delivery of CpG nucleic acids. This Cp
Systemic immunity induced in response to G nucleic acids included both humoral and cell-mediated responses to specific antigens that could not induce systemic immunity when administered alone to the mucosa. Furthermore, both CpG nucleic acids and cholera toxin (CT, a mucosal adjuvant that induces a Th2-like response) induced CTL. This was surprising because with systemic immunization, the presence of Th2-like antibodies is usually associated with CTL deficiency (Schirmeck et al., 1995). Based on the results presented herein, it is expected that immunostimulatory nucleic acids will function in a similar manner.

In addition, immunostimulatory nucleic acids induce mucus responses at both local mucosal sites (eg, lungs) and remote mucosal sites (eg, lower gastrointestinal tract). Significant levels of IgA antibodies are induced at remote mucosal sites by immunostimulatory nucleic acids. CT is generally considered to be a very effective mucosal adjuvant. As already reported (Snider, 199
5), CT mainly induces antibodies of the IgG1 isotype, which show a Th2-type response.
Conversely, immunostimulatory nucleic acids are predominantly by IgG2 antibodies, especially after boosting or 2
It is more Th1-like when two adjuvants are combined. TH1-type antibodies generally have a higher neutralizing capacity, and furthermore, the Th2 response in the lung is highly undesirable as it is associated with asthma (Kay, 1996, Hogg, 1997). Thus, the use of immunostimulatory nucleic acids as mucosal adjuvants has advantages that other mucosal adjuvants cannot achieve. The immunostimulatory nucleic acids of the invention are also useful as mucosal adjuvants that induce both systemic and mucosal immune responses.

Mucosal adjuvants called non-nucleic acid mucosal adjuvants can also be administered with immunostimulatory nucleic acids. As used herein, a non-nucleic acid mucosal adjuvant is an adjuvant other than an immunostimulatory nucleic acid that can induce a mucosal immune response in a subject when administered to a mucosal surface in combination with an antigen. Mucosal adjuvants include bacterial toxins such as cholera toxin (CT), CT derivatives (hereinafter: CT B subunit (CTB) (Wu et al., 199
8, Tochikubo et al., 1998); CTD53 (Val → Asp) (Fontan
a et al., 1995); CTK97 (Val → Lys) (Fontana et al., 1995); C
TK104 (Try → Lys) (Fontana et al., 1995); CTD53 / K63 (
Val → Asp, Ser → Lys) (Fontana et al., 1995); CTH54 (Ar
g → His) (Fontana et al., 1995); CTN107 (His → Asn) (Fo
ntana et al., 1995); CTE114 (Ser → Glu) (Fontana et al., 19)
95); CTE112K (Glu → Lys) (Yamamoto et al., 1997a); CT
S61F (Ser → Phe) (Yamamoto et al., 1997a, 1997b); CTS
106 (Pro → Lys) (Douce et al., 1997, Fontana et al., 1995);
CTK63 (Ser → Lys) (Douce et al., 1997, Fontana et al., 1995)
), (But not limited to)), closed zone toxin, zot, E. coli heat-labile enterotoxin, Labile Toxin (LT), LT derivative (hereinafter: LT B
Subunit (LTB) (Verweij et al., 1998); LT7K (Arg → Lys)
(Komase et al., 1998, Douce et al., 1995); LT61F (Ser → Phe
) (Komase et al., 1998); LT112K (Glu → Lys) (Komase et al.,
1998); LT118E (Gly → Glu) (Komase et al., 1998); LT14
6E (Arg → Glu) (Komase et al., 1998); LT192G (Arg → Gly)
(Komase et al., 1998); LTK63 (Ser → Lys) (Marchetti)
1998, Douce et al., 1997, 1998, Di Tommaso et al., 1996.
And LTR72 (Ala → Arg) (Giuliani et al., 1998) (
Pertussis toxin (PT) (Lycke et al., 1992, Span).
gler BD, 1992, Freytag and Elements, 1999, Ro
Berts et al., 1995, Wilson et al., 1995) (this is PT-9K / 129G (
Roberts et al., 1995, Cropley et al., 1995)); toxin derivatives (see below) (Holmgren et al., 1993, Verweij et al., 1998, Rappuo).
li et al., 1995, Freytag and Elements, 1999); lipid A derivatives (eg, monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vanco).
tt et al., 1998; Muranyl dipeptide (MDP) derivatives (Fukushima et al., 19
96, Ogawa et al., 1989, Michalek et al., 1983, Morisaki et al.,
1983); bacterial outer membrane proteins (eg, Borrelia burgdorferi)
Outer membrane protein A (OspA) lipoprotein, meningococcal outer membrane protein) (Mar
inaro et al., 1999, Van de Verg et al., 1996); oil-in-water emulsions (eg MF59) (Barchfield et al., 1999, Verschoor et al., 1999).
O'Hagan, 1998); aluminum salts (Isaka et al., 1998, 1999).
And saponins (eg, QS21) (Antigenics, Inc., Wobur
n, MA) (Sasaki et al., 1998, MacNeal et al., 1998), ISCOMS.
, MF-59 (Squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, CA); Monta
Sepide ISA series of nide adjuvants (e.g. Montanide
ISA720; AirLiquid, Paris, France); PROVAX (oil-in-water emulsion containing stabilizing surfactant and micelle-forming agent; IDEC Pharmaceu
ticals Corporation, San Diego, CA); Syntex
Adjuvant Formulation (SAF; Syntex Chemical)
s, Inc. , Boulder, CO); poly [di (carboxylatophenoxy) phosphazene (PCPP polymer; Virus Research Institute, US)
A) and Leishmania elongation factor (Corixa Corporation, Sea)
(ttle, WA)) (but not limited to).

The immune response is also expressed by cytokines (Bueler & Mulligan, 1996; C
how et al., 1997; Geissler et al., 1997; Iwasaki et al., 1997; K
im et al., 1997) or B-7 costimulatory molecules (Iwasaki et al .; 1997; Tsu;
ji et al., 1997) and immunostimulatory nucleic acid or co-linear
It can be induced or enhanced by expression. The cytokine can be administered directly with the immunostimulatory nucleic acid or can be administered in the form of a nucleic acid vector encoding the cytokine,
As a result, this cytokine can be expressed in vivo. In one embodiment, the cytokine is administered in the form of a plasmid expression vector. The term cytokine refers to a variety of soluble proteins and peptides that act as humoral regulators at nanomolar or picomolar concentrations and modulate the functional activity of individual cells and tissues, either under normal or physiological conditions. Used as a general name for the group. These proteins also directly mediate cell-cell interactions and regulate processes that take place in the extracellular environment.

Examples of cytokines include IL-1, IL-2, IL-4, IL-5, IL-6, I
L-7, IL-10, IL-12, IL-15, IL-18, granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), interferon-γ (γ-IFN) ), IFN-α, tumor necrosis factor (TNF), TGF-β, FLT-
3 ligands and CD40 ligands are included, but are not limited to these.

Cytokines play a role in directing T cell responses. Helper (CD4 +) T cells, through the production of soluble factors that act on other immune system cells (including other T cells)
Modulates the mammalian immune response. Most mature CD4 + T helper cells express one of two cytokine profiles (Th1 or Th2). The Th1 subset promotes delayed-type hypersensitivity, cell-mediated immunity, and immunoglobulin class switch to IgG _2a . The Th2 subset activates B cells, promotes antibody production, and IgG
Humoral immunity is induced by inducing a class switch to 1 and IgE. In some embodiments, the cytokine is preferably a Th1 cytokine.

The immunostimulatory nucleic acid can be administered directly to the subject or can be administered with the nucleic acid delivery complex. A nucleic acid delivery complex is associated with a targeting means (eg, a molecule that produces a higher binding affinity for the target cell (eg, increased cell uptake by the B cell surface and / or target cell)) (eg, Means an ionic or covalent bond; or a nucleic acid molecule encapsulated therein. Examples of nucleic acid delivery complexes include sterols (eg, cholesterol), lipids (eg, cationic lipids, virosomes or liposomes), or target cell specific binding agents (eg, ligands recognized by target cell specific receptors).
Is mentioned. Preferred complexes can be stable in vivo sufficiently to prevent significant uncoupling prior to internalization by the target cell. However, the complex may be cleavable under appropriate conditions in the cell so that the nucleic acid is released in a functional form.

A delivery vehicle or delivery device has been described for delivering antigens and nucleic acids to a surface. Immunostimulatory nucleic acids and / or antigens and / or other therapeutic agents can be administered alone (eg, in saline or in a buffer) or using any delivery vehicle known in the art. . For example, the following delivery vehicles have been described: Cochelates (C
ochleates) (Gould-Fogerite et al., 1994, 1996); Emulsomes (Vancott et al., 1998, Lowell et al., 19).
97); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu
1998, Morein et al., 1999); liposomes (Childers et al., 199).
9, Michael et al., 1989, 1992, de Haan 1995a, 1995.
b); live bacterial vectors (eg Salmonella, Escherichia)
coli, Bacillus calmatte-guerin, Shigella, L
actobacillus) (Hone et al., 1996, Pouwels et al., 1998, C
hatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998.
Live virus vectors (eg vaccinia, adenovirus, herpes simplex) (
Gallican et al., 1993, 1995, Moss et al., 1996, Nugent et al.,
1998, Flexner et al., 1988, Morrow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Malloy et al., 1994, Moo.
re et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1).
998, Okada et al., 1997, Ishii et al., 1997); polymers (eg, carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-
Gill et al., 1998); polymer rings (Wyatt et al., 1998); Proteosom
es (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997)
Sodium fluoride (Hashi et al., 1998); transgenic plants (Tacke);
t et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Glu
ck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); virus-like particles (Jiang et al., 1999, Leibl et al., 1998). Other delivery vehicles are known in the art, and some additional examples are provided below in the discussion of vectors.

The stimulation index of a particular immunostimulatory nucleic acid can be tested in various immune cell assays.
Preferably, the stimulation index of the immunostimulatory nucleic acid with respect to B cell proliferation is at least about 5, preferably at least about 10, more preferably, as determined by incorporation of ³ H uridine in mouse B cell culture. At least about 15, and most preferably at least about 20. The mouse B cell culture was contacted with 20 μM nucleic acid for 20 hours at 37 ° C. and pulsed with 1 μCi of ³ H uridine; and collected and counted after 4 hours (this was 1995, respectively) Filed on 7 February and 30 October 1997,
PCT published patent applications PCT / US95 / 01570 (WO 96/02555) and PCT / US97 / 19791 (WO 98/18810) claiming priority to US application numbers 08 / 386,063 and 08 / 960,774. ) Is described in detail). For example, it is important that an immunostimulatory nucleic acid can effectively induce an immune response (eg, antibody production) for in vivo use.

Immunostimulatory nucleic acids are effective in non-rodent vertebrates. Different immunostimulatory nucleic acids
Depending on the type of subject and the sequence of the immunostimulatory nucleic acid, optimal immune stimulation can be triggered. It has been found according to the present invention that many vertebrates respond to the same class of immunostimulatory nucleic acids (sometimes referred to as human-specific immunostimulatory nucleic acids). However, rodents respond to different nucleic acids. As shown herein, immunostimulatory nucleic acids that cause optimal stimulation in humans generally do not cause optimal stimulation in mice, and vice versa. However, immunostimulatory nucleic acids that cause optimal stimulation in humans are
Causes optimal stimulation in other animals (eg, cows, horses, sheep, etc.). Those skilled in the art can use the guidance provided herein to identify the target using conventional assays described herein and / or conventional assays known in the art. Optimal nucleic acid sequences useful for that species can be identified.

The term effective amount of an immunostimulatory nucleic acid refers to the amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of an immunostimulatory nucleic acid to induce mucinous immunity is the amount necessary to cause IgA development in response to an antigen when exposed to the antigen, whereas systemic The amount required to induce immunity is that amount necessary to cause the generation of IgG in response to the antigen when exposed to the antigen. In combination with the techniques provided herein, various active compounds are selected and factors (eg, potency, relative bioavailability, patient weight, severity of adverse side effects and preferred mode of administration) By comparing these, an effective treatment regimen or therapeutic treatment regimen that does not cause substantial toxicity and is generally effective in treating a particular subject can be planned. The effective amount for any particular application will depend on factors such as the disease or condition being treated, the particular immunostimulatory nucleic acid being administered, the antigen, the size of the subject, or the severity of the disease or condition. And can fluctuate. One skilled in the art can empirically determine the effective amount of a particular immunostimulatory nucleic acid and / or antigen and / or other therapeutic agent without necessitating undue experimentation.

Subject doses of the compounds described herein for mucosal or topical delivery typically range from about 0.1 μg to 10 mg per dose, which can be daily, weekly, or monthly and Depends on the application that can be given in any other amount of time between them. More typically, the mucosal or topical dose is 10 μg to 5 mg per administration, most typically about 10
It is in the range of 0 μg to 1 mg, and 2 to 4 doses are made at day or week intervals. More typically, the immunostimulatory dose is 1 μg to 10 mg per administration, most typically 1
Daily or weekly administration in the range of 0 μg to 1 mg. A subject dose of a compound is described herein for parenteral delivery to induce an antigen-specific immune response, where the compound is delivered with the antigen, but another therapeutic agent is Typically 5 to 10,000 times higher than the effective mucosal dose for vaccine adjuvant or immunostimulatory applications, more typically 10 to 1,000 times higher, most typically 20 ~ 100 times higher. When immunostimulatory nucleic acids are administered in combination with other therapeutic agents or in a specific delivery vehicle,
To induce an innate immune response, or to increase ADCC, or to induce an antigen-specific immune response, the doses of the compounds described herein for parenteral delivery are typically In the range of about 0.1 μg to 10 mg per dose, depending on the application that can be given daily, weekly, or monthly and any other amount of time in between. More typically, parenteral doses for these purposes range from about 10 μg to 5 mg per administration, most typically from about 100 μg to 1 mg, with 2-4 administrations per day or week Made at intervals. However, in certain embodiments, parenteral doses for these purposes can be used in the range of 5-10,000 times higher than the representative doses described above.

For any compound described herein, the therapeutically effective amount can be initially determined from animal models. Therapeutically effective doses also include compounds that are known to show similar pharmacological activity (eg, other mucosal adjuvants (such as other mucosal adjuvants)) For example, for mucosal or topical administration (for example LT and other antigens for vaccination purposes), it can first be determined from human data, higher doses are required for parenteral administration. May be adjusted based on the relative bioavailability and potency of the compound being administered, and may be adjusted to achieve the maximum potency based on the above methods and other methods well known in the art. This is well within the ability of one skilled in the art.

The formulations of the present invention are administered in pharmaceutically acceptable solutions, which are conventionally pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and necessary Depending on the, other therapeutic ingredients may be included.

For use in therapy, an effective amount of an immunostimulatory nucleic acid can be administered to a subject by any manner that delivers the nucleic acid to a consumable surface (eg, mucosa, systemic). Administering the pharmaceutical composition of the present invention may be accomplished by any means known to those skilled in the art. Preferred routes of administration include, but are not limited to, oral, parenteral, intramuscular, intranasal, intrathecal, inhalation, ocular, vaginal and rectal.

For oral administration, the compounds (ie immunostimulatory nucleic acids, antigens and other therapeutic agents) are
It can be readily formulated by combining the active compound with pharmaceutically acceptable carriers well known in the art. By such a carrier, the compound of the present invention is converted into a tablet (tab).
let, tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., may be formulated for oral consumption by the subject being treated. Pharmaceutical preparations for oral use are prepared by pulverizing the resulting mixture, if necessary, and adding the appropriate adjuvant, if desired, to obtain tablets or dragee cores. Processing the mixture,
It can be obtained as a solid excipient. Suitable excipients are in particular fillers (eg sugar (including lactose, sucrose, mannitol or sorbitol); cellulose preparations (eg corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, Methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP)
It is. If desired, disintegrating agents (eg, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (eg, sodium alginate) can be added. If desired, oral formulations can also be used to neutralize internal acid conditions. Can be formulated in saline or buffer, or can be administered without any carrier.

The dragee core is provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally include gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic A solvent or solvent mixture may be included. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules contain a filler (eg, lactose), a binder (eg, starch), and / or
Or it can be mixed with a lubricant (eg, talc or magnesium stearate) and optionally a stabilizer to contain the active ingredient. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres are well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the composition may take the form of tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compounds for use according to the present invention use a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). Can be delivered conventionally in the form of an aerosol spray presentation from a pressurized pack or nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a measured amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators are suitable powder bases such as lactose or starch
And a powder mixture.

The compounds can be formulated for parenteral administration by infusion (eg, by bolus injection or continuous infusion) if desired to be delivered systemically. Formulations for injection can be made up by adding preservatives in unit dosage forms (eg, in ampoules) or in multi-dose devices,
Can be presented. The composition may take a form such as a suspension, solution or emulsion in an oily vehicle or an aqueous vehicle. The composition can include a formulation (eg, a suspending, stabilizing, and / or dispersing agent).

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or lipophilic vehicles include fatty oils (eg, sesame oil), or synthetic fatty acid esters (eg, ethyl oleate or triglycerides), or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. If necessary,
The suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions.

Alternatively, the active compound can be in powder form for constitution prior to use with a suitable vehicle (eg, sterile pyrogen-free water).

The compounds also include, for example, conventional suppository bases (eg, cocoa butter or other glycerides) in rectal or vaginal compositions (eg, suppositories or retention enemas)
Can be prescribed.

In addition to the above formulations, the compounds can also be formulated as a depot preparation. Such long acting formulations may be prepared using suitable polymeric or hydrophobic materials (eg, as an emulsion in an acceptable oil) or ion exchange resins, or foam soluble derivatives (eg, foam dissolved). Salt).

The pharmaceutical composition may also include a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers (eg, polyethylene glycol).

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous solutions for inhalation or saline solutions, microencapsulated, swirled or coated on microscopic gold particles. Contained in liposomes, sprayed, aerosols, pellets for skin transplantation, or dried on sharp objects to be scratched into the skin. The pharmaceutical composition also comprises granules, powders, tablets, coated, (micro) capsules, suppositories, syrups, emulsions, turbids, creams, drops or preparations that release the active compound over time. Inclusion and preparation excipients and additives and / or adjuvants (eg disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners or solubilizers) as described above. Used conventionally. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533, 1990 (
See incorporated herein by reference).

The immunostimulatory nucleic acid and optionally other therapeutic agents and / or agents can be administered per se (neat) or can be administered in the form of a pharmaceutically acceptable salt. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts are conventionally used to prepare the pharmaceutically acceptable salts. obtain. Such salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid , Tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid,
Naphthalene-2-sulfonic acid, and benzenesulfonic acid. Such salts can also be prepared as alkali metal salts or alkaline earth metal salts (eg, sodium, potassium or calcium salts of carboxylic acid groups).

Suitable buffers include acetic acid and salt (1-2% w / v); citric acid and salt (1-3% w / v
); Boric acid and salt (0.5-2.5% w / v); and phosphoric acid and salt (0.8-2% w / v)
Is mentioned. Suitable preservatives include benzalkonium chloride (0.003-0.03%
w / v); chlorobutanol (0.3-0.9% w / v); paraben (0.01-0.2)
5% w / v) and thimerosal (0.004-0.02% w / v).

As described in more detail herein, the pharmaceutical composition of the present invention comprises an effective amount of an immunostimulatory nucleic acid and, optionally, an antigen and / or other therapeutic agent, optionally a pharmaceutical. In an optionally acceptable carrier. The term “pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans or other vertebrates. means. The term “carrier” means an organic or inorganic component (either natural or synthetic) into which the active ingredient is mixed for ease of application.
Means. The components of the pharmaceutical composition can also be mixed with the compounds of the present invention and mixed with each other in a manner that does not have interactions that substantially impair the desired pharmaceutical efficacy.

Immunostimulatory nucleic acids useful in the present invention can be delivered in a mixture with additional adjuvants, other therapeutic agents, or antigens. The mixture may consist of several adjuvants in addition to the immunostimulatory nucleic acid or some antigen or other therapeutic agent.

A variety of administration routes are available. The particular mode chosen will, of course, depend on the particular adjuvant or antigen chosen, the particular condition being treated, and the dosage required for therapeutic efficacy. The methods of the present invention can be performed generally using any medically acceptable mode of administration. By this mode is meant any mode that produces effective immune response levels without causing adverse effects that are not clinically acceptable. The preferred mode of administration is
Considered above.

The composition can be conventionally presented in unit dosage form and can be prepared by any of the surrounding methods in the pharmaceutical arts. All methods include the step of bringing the compounds into association with a carrier which constitutes one or more accessory ingredients. In general, the composition is prepared by uniformly and intimately associating the compound with a liquid carrier, a finely divided solid carrier, or both, and then shaping the product if necessary. . Liquid dose units are vials or ampoules. Suitable dosage units are tablets, capsules and suppositories. For patient treatment, depending on the activity of the compound, depending on the mode of administration, the purpose of immunization (ie, prophylactic or therapeutic), the nature and severity of the disorder, the age and weight of the patient, Various doses may be necessary. Administration of a given dose can be carried out both by a single dose of individual dose units or by several smaller dose units.

Other delivery systems include timed release delivery systems, delayed release delivery systems, or slow broadcast delivery systems. Such a system can avoid repeated administration of the compound, thereby increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems such as poly (lactide-glycolide), copolyoxalate, polycaprolactone, polyesteramide, polyorthoester, polyhydroxybutyric acid, and polyanhydrides.

Microcapsules of the above polymer containing a drug are described, for example, in US Pat. No. 5,075,109. Delivery systems also include non-polymeric systems, including lipids (such as sterols (eg, cholesterol, cholesterol esters), and fatty acids or natural fats (eg, monoglycerides, diglycerides, and triglycerides));
Hydrogel release systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants and the like. Specific examples include: (a) an erosion system (US Pat. Nos. 4,452,775, 4,675,189 and 5,731) wherein the agent of the present invention is included in a form in a matrix.
(B) a diffusion system (US Pat. Nos. 3,854,480, 5,133,974) and (b) in which the active ingredient penetrates at a controlled rate from the polymer. No. 5,407,686), but is not limited thereto. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

The invention is further illustrated by the following examples. The following examples should in no way be construed as further limiting. The entire contents of all references cited throughout this specification (literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. Incorporated.

(Example 1 (ODN 10102)
(Summary)
This report summarizes in vitro data using human cells. This data is ODN 1
0102 (SEQ ID NO: 1) is shown to behave similarly to ODN 7909 (SEQ ID NO: 2) and in some ways in a better manner. In vitro and in vivo data in mice show that CpG ODN 10102 enhances the innate immune system and enhances HBsAg-specific humoral and cellular responses in mice when co-administered with the antigen. , Which has similar properties as CpG ODN 7909, and sometimes better properties.

The assays performed included receptor binding (TLR9), B cell activation (expression of cell surface activation markers and B cell proliferation) and cytokine secretion (IL-10, IP-10, I
FN-α and TNF-α). All assays have ODN 10102
It was shown to have characteristics that are almost identical if not better than DN 7909.

In vitro studies (ie B cell proliferation assay, NK lytic activity, cytokine secretion profile) were performed using unstimulated BALB / c mouse splenocytes. In vivo comparative studies were performed by testing the ability of these two ODNs to enhance the antigen-specific immune response against hepatitis B antigen (HBsAg).

(Materials and methods)
(Human research)
(Oligodeoxynucleotides) All ODNs are from Coley Pharmace
provided by utical GmbH (Langenfeld, Germany). The control ODN did not contain any stimulating CpG motif. ODN was diluted in phosphate buffered saline and stored at -20 ° C. All dilutions were performed using pyrogen free reagents.

TL9 assay The cells used for this assay expressed the human TLR9 receptor and included a reporter gene construct. Cells were incubated with ODN for 16 hours. Each data point was done in triplicate. Cells were lysed and assayed for reporter gene activity. The stimulation index was calculated with reference to the reporter gene activity of the medium without the addition of ODN.

(Cell purification) Peripheral blood buffy coat preparations derived from healthy human donors were obtained from German Red C
from Ross (Rathingen, Germany), from which Ficoll-
PBMCs were purified by centrifugation against Hypaque (Sigma, Germany). The purified PBMC was used fresh or suspended in freezing media and stored at -70 ° C. If necessary, aliquots of these cells are thawed, washed, and 10% (v / v) heat-inactivated FCS, 1.5 mM L-glutamine, 100 U /
RPMI 164 supplemented with ml penicillin and 100 μg / ml streptomycin
Resuspended in 0 culture medium.

(Cytokine detection) Thawed PBMC or fresh PBMC at a concentration of 5 × 10 ⁶ /
Resuspended in ml and added to 48 well flat bottom plates (1 ml / well). Before this plate, nothing was added or various concentrations of ODN were added.
The cells were cultured at 37 ° C. in a humidified incubator. Culture supernatants were collected after the designated time points. If not used immediately, the supernatant was frozen at −20 ° C. until needed. Certain cytokines in the supernatant were purified from commercially available ELISA kits (IL-10; Diaclone
USA) or in-house ELISA (IP-19 and IFN)
-Α) and evaluated using commercially available antibodies (Pharmingen, Germany or P
Color was developed using BL, USA).

(Culture for flow cytometric analysis of B cell activation) Monoclonal antibody against CD19 and monoclonal antibody against CD86 were mixed with Becton Dicki.
Purchased from nson (Germany). PBMC were incubated for 48 hours with or without various concentrations of ODN. B cells were identified by CD19 expression by flow cytometry. Flow cytometry data is FACS
Obtained at Calibur (Becton Dickinson). Data was analyzed using the computer program CellQuest (Becton Dickinson). Proliferating CD19 positive B cells were cultured with CFSE-labeled PBMC (CFSE is a fluorescent dye that binds to all cell surfaces) and then flow cytometry methods (see above) were used for CFSE. Identified by a decrease in content.

(In vitro / in vivo studies in mice)
(Oligodeoxynucleotide) CpG ODN (GMP quality) is
hermetic Inc. (Wellsley, MA). All ODNs were sterilized without endotoxin (pH 8.0) (Omn
resuspended in iPer®; EM Science, Gibbstown, NJ), stored, and handled under aseptic conditions to prevent microbial and endotoxin contamination at the most. Dilute the assay ODN with sterile PB without endotoxin
S (pH 7.2) (Sigma Chemical Company, St. Louis)
, MO).

Animals: Female BALB / c mice (6-8 weeks old) were used for all experiments.
Animals were purchased from Charles River Canada (Quebec, Canada) and were purchased from the Ottawa Hospital Research Institute,
They were housed in micro-isolated incubators at the Civic Site Animal Care Facility.

Harvested splenocytes and cultures Unstimulated BALB / c mouse splenocytes were used for all in vitro assays. Animals were anesthetized with isoflurane and euthanized by cervical dislocation. Spleens were removed under aseptic conditions and placed in PBS + 0.2% bovine serum albumin (Sigma Chemical Company). The spleen was then homogenized and splenocytes were collected in 2% normal mouse serum (final concentration 100 U / ml; Cedarla
ne Laboratories, Ontario, Canada), penicillin-streptomycin solution (final concentration 1 mg / ml; Sigma Chemical Comp)
any) and 5 × 10 ⁻⁵ M β-mercaptoethanol (Sigma Chemical).
RPMI 1640 (Life Technology) supplemented with al Company
(gees, Grand Island, NY) Resuspended in tissue culture medium.

B cell proliferation assay: Spleen cell suspensions were prepared and adjusted to a final concentration of 5 × 10 ⁶ cells per ml in complete RPMI 1640. Splenocyte suspension is poured into 96 well U bottom tissue culture plate (100 μl / well) with 100 μl of stimulant to complete RPM
Dilute to appropriate concentration in I 1640. The stimulant used was CpG ODN (1, 3
7909 and 10102, 10103, 10104, 10105 (at 10 μg / ml)
Or 10106. Concanavalin A (10 μg / ml, Sigma Chem
ical Company) and LPS (10 μg / ml, Sigma Chemical)
al Company) was used as a positive control, and cells cultured in medium alone were used as a negative control. Each splenocyte sample was plated in triplicate and the cells were incubated at 37 ° C. for 96 hours in a humidified 5% CO ₂ incubator. At the end of the incubation period, cells were pulsed with ³ H-thymidine (20 μCi / ml) for 16 hours after 96 hours of incubation, harvested, and radioactivity measured.

Cytokine secretion profile: Spleen cell suspensions were prepared and plated into 96 well U bottom tissue culture plates as described for B cell proliferation assays. Each splenocyte sample was plated in triplicate and the cells were incubated in a humidified 5% CO ₂ incubator for 6 hours, 12 hours or 48 hours at 37 ° C. At the end of the incubation period, the 96-well plate was centrifuged at 1200 rpm for 5 minutes and the culture suspension was collected and stored at −80 ° C. until assayed. Commercially available assay kit (mouse OptEIA kit; PharMingen, Mississauga
, ON) according to the manufacturer's instructions, 6 hours (TNF-α), 24 hours (IL-1
Cytokine levels of culture suspensions removed at 2) and 48 hours (IL-6 and IL-10) were assayed.

NK assay: Spleen cell suspensions were prepared as described above and prepared to a final concentration of 3 × 10 ⁶ cells per ml in complete RPMI 1640. Spleen cell suspension (10 ml; 30 ×
10 ⁶ cells) of CpG ODN (1, 3, 10 μg / ml) 7909 and 10102,
Plated in T-25 tissue culture flasks (Fisher Scientific, Ottawa, ON) with either 10103, 10104, 10105 or 10106. Spleen cells cultured in medium alone were used as a negative control. Each splenocyte culture was incubated for 24 hours at 37 ° C. in a humidified 5% CO ₂ incubator. At the end of the incubation period, cells were plated with different effectors: 96 with 100 μl ⁵¹ Cr labeled target cells at 5 × 10 ⁴ cells / ml.
Target ratio of well U bottom tissue culture plate (100 μl / well). NK-sensitive mouse lymphocyte cell line YAC-1 (ATCC No. TIB-160, ATCC, Manassas, V
A) was used as the target cell line. Each sample was plated in triplicate and the cells were incubated in a humidified 5% CO ₂ incubator at 37 ° C. for 4 hours. Target cells were incubated with medium alone or medium containing 2N HCL to determine spontaneous and maximal release, respectively. At the end of the incubation period, the supernatant was collected and the radioactivity level was measured using a gamma counter. The% lysis was determined using the following formula:
Specific release% = (experimental release−spontaneous release) / (maximum release−spontaneous release) × 100
.

Immunization of mice: BALA / c mice (n = 10 / group) were treated with 1 μg HBsAg
Subtype ad (International Enzymes, CA) alone or 1
0 μg CpG ODN 7909 or CpG ODN 10102, 10103, 1
Immunization in combination with either 0104, 10105 or 10106. Blood was drawn from the animals and boosted 4 weeks after the primary immunization. One week after the booster, 5 animals from each group were euthanized and the spleen removed for CTL assay.

Determination of antibody response: antibodies specific to HBsAg (anti-HBs) (IgG, IgG1
And all of IgG2a), and endpoint dilution
Quantified by ELISA assay, which was performed in triplicate on samples from individual animals. The endpoint titer was defined as the highest plasma dilution that produced an absorbance (OD 450) that was two times greater than the absorbance of non-immune plasma with a cutoff value of 0.05. These were reported as group mean titers ± SEM.

Statistical analysis: Statistical analysis was performed using the InStat program (Graph PAD So
ftware, San Diego). Statistical differences between groups are shown following Student t-test (for 2 groups) or Tukey's test (for more than 2 groups) for raw or transformed data (log ₁₀ , for heterogeneous populations) 1- Assayed by factor ANOVA.

result:
TLR9 binding: A receptor for recognition of CpG sequences has recently been identified and shown to be a member of the Toll-like receptor (TLR) family (Hemmi et al., 2000). This receptor, TLR9, is readily activated by ODN containing optimal immune stimulatory CpG sequences. We incubated cell lines stably expressing human TLR9 with different concentrations of ODN 7909 and 10102 as well as control ODN (FIG. 1).

The result is that these ODNs 10102 are equal to or higher than 7909 TLR9
Is activated. Both ODNs showed dose response curves and reached maximum activity at the same concentration. The control ODN used did not induce TLR9 activity even at the highest concentration of 12 μg / ml.

Human B cells: One feature of type B ODN is their ability to activate B cells very efficiently (Krieg et al., 1995). B cell and plasma cell-like (plasma
ytoid) DC is currently the only immune cell type known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). Therefore, the inventors
The direct activity of B cells induced by DN 7909 and 10102 was measured by: a. Up-regulation of cell surface marker CD86 (FIG. 2) and b. Measurement of B cell proliferation (Figure 3). Healthy human blood donor PBMC for CD86 expression in human B cells,
Incubated with different ODN and B cell activation was measured as described in Materials and Methods.

Both results show that 10102 and 7909 are very important stimulators of human B cells (s
(timulator). FIG. 2 shows that these CpG ODNs can only stimulate B cells in vitro at a concentration of 0.4 μg / ml. About 1.
Reached 6 μg / ml. Similar results were obtained for incubation of B cell proliferation (FIG. 3), where the stimulation index reached a maximum at about 1.6 μg / ml.

Cytokine secretion: Class B ODNs lead to Th1 dominant immune responses in vivo as well as in vitro. These are typically Th1 cytokines (eg, IFN- and IFN).
It has been found that-) and Th1-related cytokines (e.g. MCP-1 and IP-10) can be induced. In addition, low secretion of pro-inflammatory cytokines IL-6 and TNF- and secretion of negative regulator IL-10 can be observed. Therefore, the inventors measured the secretion of Th1 cytokine IFN-, chemokine IP-10 and the regulator cytokine IL-10 and pro-inflammatory cytokine TNF-. FIG. 4 shows the results for experiments performed with different donors at 0.2, 0.4, 1.6 and 5 μg / ml to measure in vitro IFN-secretion.

Both CpG ODNs (7909 and 10102) induced the highest high level of IFN-α reached at 0.4 (7909) or 1.6 μg / ml (10102). However, the largest increase in IFN-α secretion was more pronounced after stimulation with 10102 compared to 7909. In contrast, control ODN induced low amounts of IFN-α starting only at 5.0 μg / ml. Furthermore, in contrast to Conotrol ODN, ODN 7909 and 10102 induced high amounts of chemokine IP-10 as shown in FIG.

A very similar experiment was performed for IL-10 secretion (Figure 6). Again, as shown above for IFN-α, both CpG ODN 7909 and 10102 show nearly identical properties, and 10102 in some cases in these assays
Work more. In comparison, control ODN induces IL-10 secretion only at the highest concentration.

As shown in FIG. 7, both ODN 7909 and 10102 and the control ODN showed a weak secretion profile of the pro-inflammatory cytokine TNF-α compared to LPS. Again, it was found that the two ODNs also have very similar properties in this assay.

In the first set of experiments, the induction of mouse spleen cell proliferation was investigated. According to the data shown in FIG. 8, CpG ODN 10102 is equally potent as CpG ODN in inducing mouse B cell proliferation at all concentrations tested.

In the next set of experiments, secretion of various cytokines was tested during incubation of spleen cells with 101012, 7909 and control 2137. According to the data shown in FIG. 9, both CpG ODN 7909 and 10102 have essentially the same potency with respect to increased cytokine secretion by mouse splenocytes.

According to the data in FIG. 10, both CpG ODN 7909 and 10102 have essentially equal potencies with respect to increased NK cell lytic activity in mouse splenocyte cultures.

According to the results of this study (FIG. 11), use of either CpG ODN 7909 or 10102 significantly increased antibody titer against HBsAg compared to antigen alone (p <0.001), but control When using ODN in combination with HBsAg,
Anti-HBs response did not increase significantly (p = 0.86).

The increase in total IgG levels is seen in C compared to when CpG ODN 10102 is used.
When pG ODN 7909 is used, there is a slight but significant increase (p = 0.04).
).

In the mouse IgG isotype, partitioning is widely used as an indicator of the nature of the immune response where a high IgG2a / IgG1 ratio indicates a Th1 biased immune response (Constant
And Bottomly, 1997). In this study, the use of CpG ODN compared to when the antigen was used alone or in combination with control ODN 2137,
IgG2a titers were significantly increased (Ag vs. 7909 or 10102 or Ag vs. Ag
P <0.001 for +2137). However, the level of IgG2a response is CpGO
Similar to either DN 7909 or 10102 when used in combination with HBsAg (p> 0.05). Thus, both CpG ODN 7909 and 10102 are equally potent with respect to their ability to induce a Th1 biased immune response, as measured by increased levels of IgG2a over IgG1.

Conclusion:
In vitro data using human cells indicated that ODN 10102 behaves similar to or better than previously identified ODN 7909 in various assays performed.

According to the results of in vivo and in vitro studies of mice, CpG ODN 101
02, when administered with an antigen, has immunopotentiating properties similar to or better than ODN 7909, both in vitro effects on inactive immune responses as well as the ability to enhance antigen-specific responses in vivo.

(Example 2 (ODN 10103))
Summary: ODN 10103 (SEQ ID NO: 19) compared its ability in vitro to stimulate human PBMC with that of ODN 7909 (SEQ ID NO: 2). Immunostimulation involves receptor (ie, TLR9) binding, B cell activation (eg, expression of cell surface activation markers and B cell proliferation) and cytokine secretion (eg, IL-10, IP-10, IFN-
Analysis of α and TNF-α secretion. All assays showed that ODN 10103 has properties similar to or better than those of ODN 7909.

The ability of ODN 10103 to stimulate mouse immune cells in vitro and in vivo,
Compared to ODN 7909. In vitro studies (eg B cell proliferation assay, NK
Lysis activity and cytokine secretion profiles) were performed using naive BALB / c mouse splenocytes. In vivo studies are performed by testing the strength of these two ODNs to enhance the antigen-specific immune response against hepatitis B surface antigen (HBsAg),
Body fluid (antibody) and cell-mediated immune response (CTL activity) were analyzed. In addition, the Th bias of the induced immune response was tested by determining the strength of the CTL response as well as the IgG2a / IgG1 ratio.

Materials and methods:
For human studies, oligodeoxynucleotides, TLR9 assay, human cell proliferation,
See Example 1 for a description of cell cultures for flow cytometric analysis of cytokine detection and B cell activation.

For in vitro and in vivo studies of mice, oligodeoxynucleotides, animals, splenocyte collection and culture, B cell proliferation assay, cytokine secretion profile, NK
See Example 1 for a description of the assay, immunization of mice, measurement of antibody response and statistical analysis.

(In vitro / in vivo study of mice :)
Evaluation of CTL response: CTl assay was performed as previously described by Davis et al. Results are presented as% specific lysis at different effector: target (E: T) ratios.

(result:)
TLR9 binding: We incubated cell lines stably expressing human TLR9 with different concentrations of ODN 7909 and 10103 and control ODN (
FIG. 13). Both ODN showed concentration-dependent dose-response curves that achieved their maximum activity at the same concentration. The control ODN is TLR9 even at the highest concentration of 24 μg / ml.
It did not induce activation. ODN 10103 exhibits higher stimulatory capacity at lower doses (eg, 6 g / ml and 12 g / ml) than does ODN 7909, which
DN 10103 can be used at lower doses to achieve a similar immune stimulation index,
It suggests that it reduces potential toxicity.

Human B cells: One property of type B ODN is their ability to activate B cells very effectively (Krieg et al., 1995). B cell and plasma cell-like (plasmacy)
toid) DC is the only immune cell type currently known to express TLR9 (Krug et al., 2001; Bauer et al., 2001). We therefore measured the direct activity of B cells induced by ODN 7909 and 10103 by up-regulation of the cell surface marker CD86 (FIG. 14) and measured the proliferation of B cells (FIG. 15). ). For CD86 expression on human B cells, healthy blood donor PBMCs were incubated with different ODNs and B cell activity was measured as described in Materials and Methods.

Both results indicate that 10103 is at least equivalent to 7909 as a stimulator of human B cells. FIG. 14 shows that these CpG ODNs can stimulate B cells starting at in vitro concentrations (only at 0.4 μg / ml). A plateau was achieved at about 1.6 μg / ml and more than 60% B cells were found to upregulate CD86 compared to a control with very little effect at the same concentration. This result indicates that ODN 10103 stimulates CD86 expression to higher levels at a lower dose (eg, 0.4 μg / ml) than the dose of ODN 7909 in all three donors tested. Again, this means that using a smaller dose of ODN 10103,
It suggests that the desired level of immune stimulation can be achieved. Similar results were obtained for induction of B cell proliferation (FIG. 15).

Cytokine secretion: Class B ODNs lead to Th1-dominant immune responses in vivo as well as in vitro. These are typically Th1 cytokines (eg, IFN-γ and I
It has been found that FN-α) and chemokines such as MCP-1 and IP-10 can be induced. Furthermore, low secretion of the pro-inflammatory cytokine Il-6 and TFN-α and secretion of the negative regulator IL-10 were observed. Therefore, we measured the secretion of Th1 cytokine IFN-α, chemokine IP-10 and regulatory cytokine Il-10 and pro-inflammatory cytokine TNF-α.

FIG. 16 shows the determination of 0.2 μg / ml, 0.1 μg to measure IFN-α secretion in vitro.
Results are shown for experiments performed with 6 different donors at 4 μg / ml and 1.6 μg / ml. Both CpG ODNs (7909 and 10103) induced significant amounts of IFN-α in different donors. In contrast, the control ODN is I
No FN-α was induced or a small amount of IFN-α was induced in one donor. This data suggests that patient variability may exist and that some patients respond better to ODNs such as 10103 when compared to ODN 7909. This finding is
4 shows that DN can be classified in terms of subjects that are likely to be high responders.

In addition to IFN-α, ODN 7909 and 10103 induced chemokine IP-10 as shown in FIG. ODN10103 is ODN7 at all doses tested
IP-10 at the same level as 909 or higher than ODN 7909 was induced. In particular, at a concentration of 0.4 μg / ml, ODN10103 produced a greater amount of IP-10 than did ODN7909. At a concentration of 1.6 μg / ml, ODN10103 is
About 25% more IP-10 was induced than N7909 induced.

As shown in FIG. 18, CpG ODN 7909 and 10103 are almost identical IL
It showed -10 inductive capacity.

As shown in FIG. 19, both ODN 7909 and 10103, as well as control ODN, showed a low secretion profile of the pro-inflammatory cytokine TNF-α at all concentrations tested compared to LPS. The highest dose tested (ie 6 μg /
ml), ODN10103 produces higher levels of IL- than ODN7909 stimulated.
Stimulated 10 secretions. The dose response is shown in FIG.

In vitro mouse studies: According to the data, both CpG ODN 7909 and 10103 are equally potent in inducing mouse B cell proliferation and have essentially the same potency in enhancing cytokine secretion by mouse splenocytes. And has essentially the same efficacy in enhancing the lytic activity of NK cells in mouse splenocyte cultures (FIG. 22). ODN 10103 appears to have a higher capacity for stimulating IL-6 and TNF- secretion, especially at the lower doses tested. Similarly, the lytic activity profile of these ODNs varies with concentration.

In vivo mouse study: According to the results of this study, CpG ODN 7909 or 101
Use of either 03 significantly enhanced antibody titer against HBsAg compared to antigen alone (p <0.001 and p <0.01, respectively), but control ODN was HB
When used in combination with sAg, there was no significant increase in anti-HBs response (p = 0.85). (See Figures 23 and 24).

Furthermore, both CpG ODN 7909 and 10103 are HBsAg + CpGO
In mice immunized with DN7909 and HBsAg + CpG ODN10103,
HBs in that there was no significant difference in anti-HBs response (p = 0.13)
Equally powerful in enhancing the antibody response to Ag.

In mouse IgG, isotype distribution is widely used as an indicator of the nature of the immune response, where a high IgG2a / IgG1 ratio indicates a Th1 biased immune response (1).
In this study, the use of CpG ODN significantly enhanced IgG2a titers (Ag vs. 7909 or Ag vs. 10103) compared to when the antigen was used alone or in combination with the control ODN2137. P <0.001
And p + 0.01 for Ag + 7909 vs. Ag + 2137, and Ag + 10
103 vs. Ag + 2137, p <0.05). However, the level of IgG2a response was similar when either CpG ODN 7909 or 10103 was used in combination with HBsAg (p> 0.05). Thus, both CpG ODN 7909 and 10103 are equally potent in their ability to induce a Th1-biased immune response as measured by increased levels of IgG2a against IgG1.

As shown in FIG. 25, the CTL response in animals immunized with HBsAg using ODN10103 appears to be greater than the CTL response that induced CpG ODN7909.

Conclusion: In vitro data in human PBMC demonstrates that in all assays performed, B-class molecules (7909 and 10103) behave similarly but are not identical. Of particular importance is the OD for some of the assays and correlations tested.
It is an observation that N10103 induced greater immune stimulation than the previously identified ODN 7909 induced. This difference suggests that CpG nucleotides can be tailored for use in subjects and that lower levels of CpG ODN can achieve the desired therapeutic endpoint with potentially lower toxic events.

(Example 3 (ODN 10104)):
(In vivo effect):
Overview:
Synthetic oligodeoxynucleotides containing non-methylated CpG dinucleotides (O
DN) has been shown to induce a strong innate immune response against infectious agents and cause Th1-like immune activation. Transmucosal of CpG ODN applied to genital mucosa
cosal) delivery was tested for its ability to protect against or treat intravaginal (IVAG) infection with herpes simplex virus type 2 (HSV-2).

Materials and methods:
All ODNs are available from the Coley Pharmaceutical Group (Lan
Genfeld, Germany) and the Limulus assay (Bio
Had undetectable endotoxin levels (<0.1 EU / ml) measured by Whittaker, Verviers, Belgium). ODN was suspended in sterile endotoxin-free Tris-EDTA (Sigma, Deisenhofen, Germany) and stored and processed under aseptic conditions to prevent both microbial and endotoxin contamination. All dilutions were pyrogen-free phosphate buffered saline (Life Tech).
nologies, Eggenstein, Germany).

CpG ODN and non-CpG ODN may be pre-IVAG HSV-2 infection or IVA
At various times after GHSV-2 infection, it was applied to the vagina of female C57B1 / 6 mice. Mice were monitored daily for genital pathology, survival, and genital virus titers after challenge.

result:
Female mice treated with CpG ODN in the genital tract 24 hours prior to IVAG HSV-2 challenge survived the infection, showed minimal genital pathology, and substantially vaginal lavage fluid for the first 6 days after infection. There was no virus inside. Importantly,
Transmucosal delivery of CpG ODN to the genital tract prior to infection provided superior protection against local IVAG challenge compared to intramuscular delivery of CpG ODN. In contrast, Control O
Mice pretreated with DN alone were not protected, showed severe pathology, and had high titers of HSV-2 in the vaginal lavage fluid. Mice treated with CpG ODN immediately after IVAG HSV-2 infection were protected and had low vaginal virus titers, while mice treated with CpG ODN were protected 24 and 72 hours after IVAG HSV-2 infection. None (see FIGS. 26 and 27).

Conclusion:
These results indicate that CpG into the genital tract before or immediately after the genital HSV-2 challenge
We show that local transmucosal delivery of ODN was very effective in preventing infection by sexually transmitted viruses and suggest that local CpG-induced innate immunity is involved.

(In vitro effect):
Materials and methods:
Peripheral blood buffy coat preparations from healthy human donors were purchased from German Red Cross (
Obtained from Rathingen, Germany) and from these, PBMCs were obtained from Fico
Purified by centrifugation against ll-Hypaque (Sigma, Germany). Purified PBMC were either used as is or suspended in frozen media and stored at -70 ° C. If necessary, aliquots of these cells are thawed, washed and 10% (v / v) heat inactivated FCS, 1.5 mM L-glutamine, 100 U.
RPMI 16 supplemented with / ml penicillin and 100 μg / ml streptomycin
Resuspended in 40 culture medium.

Thawed PBMC or neat PBMC is resuspended at a concentration of 5 × 10 ⁶ / ml and added to a 48-well flat bottom plate that has not been given anything before or received various concentrations of ODN (1 ml / ml). Well). The cells were cultured at 37 ° C. in a humidified incubator. The culture supernatant was collected after 48 hours. If not used immediately, the supernatant was frozen at -20 ° C until needed. The amount of cytokine in the supernatant was determined using commercially available antibodies (respectively Pha
Commercially available ELISA kit (IL-10; Diaclone, USA) or in-house ELISA using rmingen or PBL; Germany or USA)
A (IFN-α) was used for evaluation.

result:
B class ODNs lead to Th1-dominant immune responses in vivo as well as in vitro. They have found that they can induce representative Th1 cytokines (eg, IFN-α and IFN-γ) and Th1-related chemokines (eg, MCP-1 and IP-10). Furthermore, low secretion of the pro-inflammatory cytokines IL-6 and TNF-α, as well as secretion of the negative regulator IL-10 can be observed. Therefore, we measured the secretion of Th1 cytokine IFN-α and the regulatory cytokine IL-10. FIG. 28 shows 0.02 μg / ml to measure IFN-α secretion in vitro,
0.05 μg / ml, 0.1 μg / ml, 0.2 μg / ml, 0.5 μg / ml and 1
. Results are shown for experiments performed with 0 μg / ml of 10104 or control nucleic acid using 3 different donors.

Nucleic acid 10104 induced high levels of IFN-α in a dose dependent manner with peak induction at 0.1 μg / ml of 10104 nucleic acid. In contrast, the control nucleic acid induced a low amount of IFN-α, which was comparable to the amount of IFN-α induced in the presence of medium alone (FIG. 28).

A similar experiment was performed for IL-10 secretion (Figure 4). Again, IFN-
As indicated above for α, nucleic acid 10104 induced IL-10 in a dose-dependent manner with peak induction at 0.2 μg / ml of 10104 nucleic acid. The control nucleic acid showed a similar but lower induction profile, but its peak shifted to 0.5 μg / ml control nucleic acid. In this case, the control level was greater than the media level (FIG. 29).

(TLR9 involvement):
Materials and methods:
Stablely transfected HEK293 cells expressing human TLR9 have been previously described (Bauer et al .; PNAS; 2001). In short, HEK293 cells
It was transfected by electroporation with a vector expressing human TLR9 and a 6xNFκB luciferase reporter plasmid. Stable transfectants (3 × 10 ⁴ cells / well) were incubated with ODN for 16 hours at 37 ° C. in a humidified incubator. Each data point was performed in triplicate. Cells are lysed and B from Perkin-Elmer (Uberlingen, Germany)
assayed for luciferase gene activity (using a rightlite kit). The stimulation index was calculated with respect to the reporter gene activity of the medium without the addition of ODN.

result:
Recently, receptors for recognition of CpG sequences have been identified and shown to be members of the Toll-like receptor (TLR) family (Hemmi et al., 2000). This receptor TLR9 is readily activated by ODN containing optimal immunostimulatory CpG sequences. We have established cell lines that stably express human TLR9 at various concentrations of 101.
Incubated with 04 and control ODN (Figure 30).

This result demonstrates that 10104 ODN activated TLR9 in a dose-dependent manner with maximal stimulation at 0.625 μg / ml. On the other hand, the control ODN only stimulated at 10 μg / ml, and the stimulation was much lower than that observed with 10104 nucleic acid.

(CpG10104 effect unrelated to delivery vehicle):
FIGS. 61A and 61B show the effect of local CpG delivery using BEMA discs or local CpG delivery in saline after intravaginal challenge with HSV-2. 2 mg / mouse to female C57 / B16 mice
SC was injected with a progesterone (4 days before virus challenge). CpG10104 (1 mg, 10 mg or 100 mg) or bioerodible (bio-
erodiable) CpG10104 impregnated on mucoadhesive disc (BEMA)
1 mg, 10 mg or 100 mg) in the vaginal cavity (I
VAG) was instilled. Using a cotton applicator on mice for virus challenge I
104 PFU for 1 hour with VAG applied, lying on top and halothane anesthesia
Infections were by instillation of 10 ml IVAG containing HSV-2 (strain 333). Genital pathology was monitored daily after HSV-2 challenge and scoring was performed in a blinded manner. Pathology was recorded on a 5-point scale: 0, no clear infection; 1, slight redness of the external vagina; 2, redness and swelling of the external vagina; 3, severe redness and swelling of the external vagina and surrounding tissues ; 4
Genital ulceration with severe redness, swelling and hair loss in the reproductive and surrounding tissues; 5, severe genital ulceration over the surrounding tissues. Mice were sacrificed when stage 5 was reached. Graph shows mean pathological value for time (days) after infection for CpG in BEMA disc (FIG. 61A) or mean pathology value for time (days) after infection for CpG in saline (FIG. 61B). Indicates.

This result shows that CpG10104 in saline or CpG10 on a BEMA disk
104 shows that IVAG delivery to mice can reduce vaginal pathology associated with subsequent IVAG HSV-2 infection.

FIGS. 62A and 62B show the effect of local CpG delivery using BEMA disc on the survival rate of mice after intravaginal challenge with HSV-2, or local C in saline.
The effect of pG delivery is shown. Mice were treated essentially as described above. Mice were monitored daily and sacrificed when severe genital ulceration extending to surrounding tissues was observed. The graph is B
% Survival with respect to time (days) after infection for CpG in EMA discs (Figure 62)
A) or% survival (Figure 62B) with respect to time (days) after infection for CpG in saline.

This result shows that CpG10104 in saline or CpG10 on a BEMA disk
104 demonstrates that IVAG delivery to mice can enhance survival after subsequent IVAG HSV-2 infection.

(Parental administration of CpG10104):
Figures 63 and 64 show the effect of parenteral CpG10104 delivery on IP-10 and IFN-γ levels in mouse plasma. 10 female BALB / c mice
SCs were injected with CpG10104 in 0 nmol saline, CpG7909 in saline, or resiquimod (R-848). Various time points after injection (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours,
10 hours, 11 hours, 12 hours), blood was collected from the mice, plasma was collected, and IP-10 levels were determined by ELISA.

This result indicates that CpG10104 can induce significant amounts of IP-10 and IFN-γ in plasma after SC injection, and the achieved levels are greater than those at R-848.

(Mucosal administration of CpG10104):
FIG. 65 shows the effect of intravaginal CpG10104 delivery on IP-10 levels in mouse plasma. Female BALB / c mice were injected into the vaginal cavity with 100 pmoles of CpG10104 in saline, CpG7909 in saline, or regiquimod (R-84).
8) was given. At various time points after injection (1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours) Plasma was collected and IP-10 levels were determined by ELISA. This result shows that CpG10
104 shows that significant amounts of IP-10 can be induced in plasma after IVAG instillation.

FIG. 69 shows the effect of intravaginal CpG10104 delivery on IP-10 levels in mouse vaginal lavage fluid. Female BALB / c mice were injected into the vaginal cavity with CpG10104 in 100 nmol saline, CpG7909 in saline, or regiquimod (R-
848). Various time points after injection (15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours
5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours)
The vaginal cavity of the mouse was washed 3 times with 75 ml of PBS. EL-10 level in vaginal lavage fluid
Determined by ISA. This result indicates that CpG10104 can induce significant local production of IP-10 in the vaginal cavity after IVAG instillation.

(Topical administration of CpG 10104)
FIG. 67 shows the effect of local CpG delivery on the local pathology of mice after intravaginal exposure with HSV-2. Female C57 / B16 mice were injected with 2 mg of progesterone SC per mouse (4 days before virus exposure). 24 hours prior to virus exposure
pG10104 (1, 10 or 100 mg) or Resquimod (1, 10
Or 100 mg) was injected into the vaginal cavity (IVAG). For viral exposure, while maintaining halothane anesthesia for 1 hour, wipe the mouse IVAG with a cotton applicator, place the mouse on its back, and 10 ml containing 104 PFU HSV-2 (strain 333) for 1 hour. Mice were infected by multiple IVAG injections. After HSV-2 exposure, genital pathology was monitored daily and scoring was performed blindly. Pathology: 0, no infection seen; 1, mild redness of external vagina; 2, redness and swelling of external vagina; 3, severe redness and swelling of external vagina and surrounding tissues; 4, genitals and surrounding tissues Genital ulceration with severe redness, swelling and hair loss; 5; scored in 5 stages of severe genital ulceration extending to surrounding tissues. When stage 5 was reached, the mice were sacrificed.

This graph shows the average pathology score versus time (days) after infection for CpG ODN 10104 or Resiquimod in saline. This result indicates that IVAG delivery of CpG 10104 to mice can reduce vaginal pathology associated with subsequent IVAG HSV-2 infection, and CpG 10104 may be more potent than a 10-fold dose of R-848 Explain that.

FIG. 68 shows the effect of topical CpG delivery on mouse survival following intravaginal exposure to HSV-2. Essentially, mice were treated as described above. Mice were monitored daily and were sacrificed when severe genital ulceration occurred to the surrounding tissues. Graph shows C in physiological saline
For pG ODN 10104 and Requiquimod,% survival versus time (days) after infection is shown.

This result shows that IVAG delivery of CpG 10104 to mice resulted in subsequent IV
Can increase survival after AG HSV-2 infection, and CpG 10104 is R-84
Explain that there may be higher potency than a 10-fold dose of 8.

FIGS. 70A and 70B show the effect of local CpG delivery on survival and local pathology of mice after intravaginal exposure of HSV-2. Essentially, mice were treated as described above. CpG 10104 (100 mg) in saline or oil-in-water cream was infused into the vaginal cavity (IVAG) either single application 4 hours after viral infection or multiple applications once a day for 5 days .
After HSV-2 exposure, genital pathology was monitored daily and scoring was performed blindly. Pathology: 0, no infection seen; 1, mild redness of external vagina; 2, redness and swelling of external vagina;
3. Severe redness and swelling of the external vagina and surrounding tissues; 4. Genital ulceration with severe redness, swelling and hair loss of the genital and surrounding tissues; Scored. When stage 5 was reached, the mice were sacrificed. This graph shows% survival versus time (days) post infection for CpG 10104 in saline (FIG. 70A
) And local pathology score (FIG. 70B).

This result explains that IVAG delivery of CpG 10104 in saline or in oil-in-water cream may reduce the pathology of mice pre-infected with lethal doses of HSV-2 and increase survival.

(Mouse immunity)
71A and 71B show that CpG 10104 is as good as CpG 7909 in promoting a humoral immune response against HBsAg in BALB / c mice. BALB / c mice were treated with HBsAg 1 mg alone or ODN 10 m
Immunized by intramuscular injection into the left anterior tibial muscle with g and / or alum (25 mg AL3 +). Animals were boosted 4 weeks after the first immunization. Antibody titers were determined 2 weeks after boost using an endpoint ELISA. FIG. 71A shows an experiment conducted without alum, and FIG. 71B shows an experiment conducted with alum.

72A and 72B show that when promoting a Th1-induced immune response (determined by high IgG2a compared to IgG1 titer) against HBsAg in BALB / c mice,
It shows that CpG 10104 is as good as CpG 7909. BALB / c
Mice were immunized by intramuscular injection into the left anterior tibialis muscle with 1 mg HBsAg alone or with 10 mg ODN and / or alum (25 mg AL3 +). 4 after the first immunization
Weekly animals were used as booths. 2 weeks after boost using endpoint ELISA,
IgG isotype levels were determined.

(Example 4 (ODN 10105))
(Summary):
This report summarizes in vitro data using human cells and explains that in human cell assays, ODN 10105 performs as well or better than ODN 7909. In addition, ODN 10105 shows similar or better performance than ODN 7909 in mouse in vitro and in vivo data.
Explain that G ODN 10105 is useful to activate the innate immune system and enhance humoral and cellular HBsAg-specific responses in mice co-administered with antigen.

The assays performed were receptor binding (TLR9), B cell activation (expression of cell surface activation markers and B cell proliferation) and cytokine secretion (IL-10, IP-10 and IFN-α). All assays were performed using ODN 10105 but ODN 790.
It has been described that it has the same characteristics as 9 or better than ODN 7909. In vitro studies (ie, B cell proliferation assay, NK lytic activity, cytokine secretion profile) were performed using naive BALB / c mouse splenocytes. In vivo comparative studies were performed by comparing the potential of these two ODNs to enhance the antigen-specific immune response against hepatitis B antigen (HBsAg).

(Materials and methods):
For human studies, oligodeoxynucleotides, TLR9 assay, human cell purification,
See Example 1 for a description of cultures for flow cytometric analysis of cytokine detection and B cell activation.

Examples for description of oligodeoxynucleotides, animals, splenocyte collection and culture, B cell proliferation assay, cytokine secretion profile, NK assay, mouse immunity, antibody response determination and statistical analysis for mice in in vitro and in vivo studies See 1.

(result):
(TLR9 binding): Cell lines that stably express human TLR9 were treated with different concentrations of ODN.
Incubated with 7909 and ODN 10105 and control ODN (Figure 31). This result explains that there was no statistically significant difference between the two B class ODNs when TLR9 was activated. Both ODNs showed the same dose-response curve and reached maximum activation at the same concentration. The control ODN used did not induce TLR9 activation even at the highest concentration of 24 μg / ml.

Human B cells: One feature of type B ODN is the ability to activate B cells very effectively (Krieg et al., 1995). B cells and plasmacytoids
oid) DC is currently the only immune cell type known to express TLR9 (
Krug et al., 2001; Bauer et al., 2001). ODN 7909 and ODN 1
The direct activity of B cells induced by 0105 is upregulated by the cell surface marker CD86 (
Figure 32) and B cell proliferation (Figure 33). For CD86 expression of human B cells, PBMCs from healthy blood donors were incubated with different ODNs and B cell activity was measured as described in Materials and Methods.

This result explains that ODN 10105 as well as ODN 7909 are extremely potent human B cell stimulators. FIG. 32 shows that these CpG ODNs were able to stimulate B cells in vitro only at a concentration of 0.4 μg / ml. 1.6 μg /
A plateau was reached in ml, and more than 70% of the B cells were found to be up-regulated, as opposed to a much less potent control. ODN 10105
Even the maximum test dose of 6 μg / ml was able to induce B cell proliferation, whereas ODN
Similar results were obtained for induction of B cell proliferation except that 7909 reached a plateau at a 1.6-3.0 μg / ml dose (FIG. 33).

Cytokine secretion: B class ODNs are Th in vivo as well as in vitro.
Induces a dominant immune response. They are representative Th1 cytokines (eg IFN
-Γ and IFN-α) and chemokines (eg MCP-1 and IP-10) were found to be able to be induced. In addition, the proinflammatory cytokines IL-6 and T
Low secretion of NF-α and negative regulator IL-10 can be observed. ODN 10105
And ODN 7909 followed by Th1 cytokine IFN-α, chemokine IP-
10 and the secretion of the regulatory cytokine IL-10 and the proinflammatory cytokine TNF-α were measured. FIG. 34 shows 0.2, 0.4 to measure IFN-α secretion in vitro.
Results of experiments performed with 6 different donors, and 1.6 μg / ml are shown.

Both CpG ODNs have high levels of INF − maximal at 0.4-1.6 μg / ml.
α was induced. In contrast, the control ODN induced a low amount of INF-α that began to express only at 5.0 μg / ml. Compared to ODN 7909, ODN 10105 induced higher levels than INF-α at both 1.6 and 5.0 μg / ml doses. In contrast to control ODN, ODN 7909 and ODN 10
105 induces chemokine IP-10 as shown in FIG. 35, again in this case ODN
10105 induced higher levels at a 0.4 μg / ml dose.

Time-dependent effects on different cytokines were also analyzed. Therefore, PBMCs from different donors were incubated for 8, 24, 36 and / or 48 hours to measure IL-10 or IFN-α secretion. FIG. 36 and FIG.
Incubate for 8 and 24 hours with two different donors (Figure 36) or 36
The results obtained for IFN-α when incubated for 48 hours and 48 hours (FIG. 37) are described. When incubated with CpG ODN, INF-α is first secreted as early as 8 hours and reaches a maximum amount at 24 hours, which remains at this level between 24 and 48 hours. Or even increased. LPS did not induce IFN-α at all. For both donors, ODN 10105 stimulated higher levels of IFN-α at 8 hours at a concentration of 1.6 μg / ml.

A very similar experiment was performed for IL-10 secretion (FIGS. 38 and 39). This cytokine showed similar characteristics to INF-α, but the maximum amount was obtained at 48 hours. Again, as described above for INF-α, CpG ODN

Both 7909 and ODN 10105 showed almost identical properties in these assays as did all other assays performed.

(In vitro mouse studies): As shown in FIG. 40, ODN 10105
It is possible to stimulate higher levels of B cell proliferation than ODN 7909 at all test concentrations. According to the data shown in FIG. 41, CpG ODN 7909 and ODN 1010
Both 5 can stimulate IL-10, IL-12, IL-6 and TNF- secretion. For IL-12 and TNF- secretion, ODN 10105 induces more factor secretion than 7909 at all test concentrations.

CpG ODN has essentially the same potency in enhancing lytic activity of NK cells in mouse splenocyte cultures (FIG. 42).

As shown in FIG. 43, either CpG ODN 7909 or ODN 10105 significantly increased the antibody titer against HBsAg compared to the antigen alone (p
<0.0001), whereas when control ODN was used in combination with HBsAg, there was no significant increase in anti-HBs response (p = 0.85).

As shown in FIG. 44, the increase in total IgG levels is similar for both CpG ODNs. In mice, IgG isotype distribution is widely used as an indicator of the nature of the immune response, where high levels of IgG2a / IgG1 indicate a Th1 biased immune response (Cons
tant and Bottomory, 1997). In the current study, the use of CpG ODN significantly increased IgG2a titers compared to when antigen alone was used or in combination with control ODN 2137 (Ag vs. ODN 790
P <0.001 for 9 or 10105 and Ag + 7909 vs. Ag + 2137
P <0.01 and Ag + 10105 versus Ag + 2137 for p <0.0
5). However, either CpG ODN 7909 or 10105 is replaced with HBsA
When used in combination with g, IgG2a response levels were similar (p> 0.05). Therefore, both CpG ODN 7909 and 10105 are IgG against IgG1.
Similarly strong in the ability to induce a Th1-biased immune response as measured by an increase level of 2.

(Conclusion):
ODN 10105 shows in vitro data using human cells, indicating that it behaves similarly to ODN 7909 and in some cases better. According to the results of mouse studies, CpG ODN 7909 and 10105 are similar for both the effect on the innate immune response in vitro as well as the ability to enhance the antigen-specific response in vivo when administered together with the antigen. Have enhanced immune enhancing properties.

(Example 5 (ODN 10106)):
(HCV research):
(Summary):
This experiment was performed using CpG ODN 10106 and CpG ODN 7909 in PBMCs isolated from normal healthy adult subjects and chronically HCV-infected adult subjects.
Were compared for their immune activity properties. The ability of ODN to stimulate B cell proliferation, cytokine secretion (IL-10 and TFN-α) and chemokine secretion (IP-10) was evaluated. All assays showed that ODN 10106 had almost the same or better properties than ODN 7909, and similar results were normal, healthy adult subjects and chronically infected with HCV PBM isolated from adult subjects
Observed with C.

(Materials and methods):
(Human (HCV) study):
(Oligodeoxynucleotide): CpG ODN 7909, 10106 and control ODN 4010 were combined with Coley Pharmaceutical Group
Made by contracting with p. Total ODN is sterilized endotoxin-free TE (pH 8.0)
^{(OmniPer R; EM Science, Gibbstown} , NJ) and resuspended in and stored and manipulated in sterile conditions in order to prevent the contamination of both microbial and endotoxin. Control ODN 4010 does not contain a stimulating CpG motif. Dilution of ODN was performed immediately before use with 10% normal human AB serum (Wisent Inc, St. Bruno,
QC) (heat inactivated) and 1% penicillin / streptomycin (Gibco BRL,
(Gland Island, NY) in RPMI 1640 complete medium. The ODN sequences used are shown in the table below.

  Table 1: ODN sequences used in these experiments.

PBMC isolation: Whole blood 2 from 10 normal healthy adult subjects and 10 adult subjects chronically infected with HCV who had failed the previous 6 months of treatment with IFN-α based treatment
00 mL was collected in a green top vacutainer supplemented with heparin by venipuncture.
Peripheral blood mononuclear cells (PBMC) were purified by centrifugation against Ficoll-Pacque at 400 × g for 35 minutes. Cells were resuspended at a concentration of 10 × 10 ⁶ / ml in RPMI complete medium containing 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin.

B cell proliferation assay: ODN in RPMI medium containing 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin at the following concentrations: 2 μg / ml, 6 μm.
Diluted to g / ml as well as 12 μg / ml. 100 μL of diluted ODN was added to the wells of a 96 well round bottom plate. Freshly isolated PBMCs are resuspended to 1 × 10 ⁶ / ml in complete RPMI medium containing 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin, then cell suspension 1 μg / mL, 3 μg /
Each well (100 μL / well) to a final ODN concentration of mL and 6 μg / mL
Added to. Cells were cultured for 5 days and then pulsed with ³ H-thymidine (1 μCi / well) for 16-18 hours. Following incubation, cells were collected on filter paper and the amount of radioactivity was measured. Results were reported as stimulation index (SI) relative to untreated media control.

Cytokine detection: ODN in RPMI medium containing 10% normal human AB serum (heat inactivated) and 1% penicillin / streptomycin at the following concentrations: 2 μg / ml, 6 μm.
Diluted to g / ml as well as 12 μg / ml. 100 μL of diluted ODN was added to the wells of a 96 well flat bottom plate. Freshly isolated PBMC resuspended at a concentration of 10 × 10 ⁶ / ml was added to each well (100 μL / well) to a final ODN concentration of 1 μg / mL, 3 μg / mL and 6 μg / mL. . Cells are 37 ° C. with 5% CO ₂
And incubated for 48 hours. Following incubation, cell supernatants were collected from each well and frozen at −80 ° C. until assayed.

The levels of IFNα and IL-10 and IP-10 in the supernatant were determined by R & D Syst
Measurements were made using ELISA Kits (catalog numbers 41105, D1000 and DIP100, respectively) commercially available from ems, Minneapolis, MN based on manufacturer's instructions.

Statistical analysis: Statistical analysis was performed using InStat (Graph PAD Software, Sa
n Diego). Statistical differences between groups were determined by one-way analysis of variance (ANOVA) on raw or transformed data (log ₁₀ ) followed by Tukey-Kramer multiple comparison test. Bartlett's test (K) indicating that the difference between the standard deviations is a significant nonparametric ANOVA when the data is transformed
urskal-Wallis test) was used.

(result)
B cell proliferation: One characteristic of type B ODN is their ability to activate B cells very efficiently (Krieg et al., 1995). Two B-class ODNs (7909 and 1
The ability of 0106) to stimulate B cell proliferation is shown in FIG. 45 below.

When compared to CpG ODN 7909, 10106 was equally effective in stimulating B cell proliferation. Furthermore, there was no significant difference in their ability to stimulate PBMC from either groups of normal healthy subjects or subjects chronically infected with HCV.

Cytokine / chemokine secretion: B class ODNs are expressed in vivo and in vitro by T
hl produces a dominant immune response. It has been found that B class ODNs can induce representative Thl cytokines such as IFNα and IFN-α, and chemokines such as MCP-1 and IP-10. In addition, the pro-inflammatory cytokine IL-
Low secretion of 6 and TNF-α, and secretion of the negative regulator IL-10
Can be observed. 46, 47, and 48 illustrate the ability of B class ODN to stimulate secretion of Thl cytokine IFNα, chemokine IP-10 and regulatory cytokine IL-10.

B class ODN (7909 and 10106) induced secretion of equivalent concentrations of IFNα.

After stimulation of PBMC with either 7909 or 10106 B-class ODN, equivalent concentrations of IP-10 were secreted. PBMC isolated from normal and healthy subjects
Or IP-10 from PBMC isolated from a subject chronically infected with HCV
There was no difference in the ability of these ODNs to stimulate the secretion of. The maximum concentration of IP-10 was obtained at an ODN concentration of 3 μg / ml for both 7909 and 10106. The same analysis was performed for IL-10 secretion (Figure 48).

CpG ODN 7909 and 10106 could cause the secretion of comparable concentrations of IL-10 from PBMC isolated from both adult populations. Maximum IL- for both ODNs
Ten inductions were observed at 6 μg / ml.

(Conclusion)
(1) normal and healthy subjects and (2) chronically HCV that previously failed IFN-α treatment
In vitro data using human peripheral blood mononuclear cells isolated from two different adult populations of subjects infected with BCl showed that B class CpG ODN 7909 and 10106 showed B cell proliferation and IFN-α in the same population. It is equally possible to stimulate the secretion of IL-10 and IP-10, and further indicates that this effect was the same for the two populations.

(Non-HCV research)
(wrap up)
CpG ODN 10106 is a class B nucleic acid. These experiments are based on CpG O
Compare the immune activation properties of DN 10106 with those of CpG ODN 7909. In vitro and in vivo immunological parameters were used for this evaluation.

In vitro data shows ODN 10106 and ODN 79 for human PBMC
It was obtained by comparing with 09. The assay performed was performed using receptor binding (TLR).
9), including B cell activation (expression of cell surface activation markers and B cell proliferation) and cytokine secretion (IL-10, IP-10, IFN-α and TNF-α). All assays showed that ODN 10106 has properties that are almost identical to ODN 7909.

In vitro studies (ie B cell proliferation assay, NK lytic activity, cytokine secretion profile) were also performed by using splenocytes of naive BALB / c mice. In vivo comparative studies were performed by examining the ability of these two ODNs to enhance antigen-specific immune responses against hepatitis B antigen (HBsAg). In in vivo comparative studies, the improvement of both humoral responses (antibodies) and cell-mediated immune responses (CTL activity) was examined. Furthermore, the nature of the immune response elicited (ie Thl vs. Th2)
The ratio was determined by determining the ratio of a / IgG1 and the strength of the CTL response.

(Materials and methods)
For human studies, see Example 1 for a description of cultures for oligodeoxynucleotides, TLR9 assay, human cell purification, cytokine detection, and flow cytometric analysis of B cell activation.

For in vitro and in vivo studies of mice, oligodeoxynucleotides,
Animal, splenocyte collection and culture, B cell proliferation analysis, cytokine secretion profile, NK
See Example 1 for a description of the assay, immunization of mice, quantification of antibody response, and statistical analysis.

(result)
(TLR9 binding)
We have established cell lines that stably express human TLR9 at different concentrations of ODN 79.
Incubated with 09 and ODN 10106, and control ODN (FIG. 49). This result shows that with respect to activating TLR9, two B-class ODNs
It shows that there was no significant difference between. Both ODNs showed the same dose response curve. The control ODN used was TLR9 even at the highest concentration of 12 μg / ml.
It did not induce activity.

(Activation of human B cells)
One feature of type B ODNs is their ability to activate B cells very efficiently (Krieg et al., 1995). Currently, only B cells and plasma cell-like DCs are
An immune cell type known to express TLR9 (Krug et al., 2001; Ba
uer et al., 2001). Accordingly, we have developed ODN 7909 and ODN 10
The direct activation of B cells induced by 106 was measured by up-regulation of the cell surface marker CD86 (FIG. 50) and by proliferation of B cells (FIG. 51). Separate the OD of healthy blood donor PBMC for CD86 expression on human B cells
Incubated with N and B cell activation was measured as described in Materials and Methods.

(B cell proliferation)
Both results indicate that 10106 and 7909 are highly potent human B cell stimulators. FIG. 50 shows that these CpG ODNs were able to stimulate B cells very potently at an in vitro concentration of only 0.4 μg / ml. The plateau has about 1.6
Reached at μg / ml. Similar results were obtained for induction of B cell proliferation (FIG. 51), where the stimulation index reached a maximum at about 0.8 μg / ml.

(Cytokine secretion)
B class ODNs produce a Th1 dominant immune response in vivo and in vitro. These can induce representative Th1 cytokines (eg, IFN-γ and IFN-α), as well as chemokines (eg, MCP-1 and IP-10). In addition, low secretion of pro-inflammatory cytokines IL-6 and TNF-α, and negative regulator IL-
Ten secretions can be observed. The investigators therefore have the Th1 cytokine IFN-α
, Chemokine IP-10, and regulator IL-10 and pro-inflammatory cytokine T
NF-α secretion was measured.

FIG. 52 shows 0.2 μg / ml, 0. 1 to measure IFN-α secretion in vitro.
Results of experiments performed with 3 different donors at 4 μg / ml, 1.6 μg / ml, and 5 μg / ml are shown. Both CpG ODNs (7909 and 10106)
Induces high levels of IFN-α, 0.4 μg / ml (7909) or 1.6 μg / m
The maximum was reached at l (10106). However, the maximum increase in IFN-α secretion appeared more than about 3 times after 10106 stimulation compared to 7909.

In addition, ODN 7909 and ODN 10106 induced a larger amount of cytokine IP-10, as shown in FIG. 53, in contrast to the control ODN. In this experiment, a plateau was already reached at about 0.2 μg / ml.

A very similar experiment was performed for IL-10 secretion (FIG. 54). Again,
CpG ODN 7909 and ODN as described above for IFN-α
Both 10106, like all other assays performed, showed nearly identical properties in this assay. In comparison, the control ODN is IL-1 only at the highest concentration.
Induces zero secretion.

As shown in FIG. 55, both ODN 7909 and ODN 10106, as well as control ODN, showed a low secretion profile of the pro-inflammatory cytokine TNF-α at all tested concentrations compared to LPS. Again, these two ODs
Similar features can be observed after stimulation with N.

According to this data, both CpG ODN 7909 and ODN 10106 are
Has substantially equivalent potency to enhanced cytokine secretion by mouse splenocytes (FIG. 57).

(B cell proliferation)
According to the data, CpG ODN 10106 is equally potent, if not above CpG ODN 7909, at induction of mouse B cell proliferation at all concentrations tested (FIG. 56).

(NK assay)
According to the data, both CpG ODN 7909 and ODN 10106 have substantially equivalent potency for enhancing NK cell lytic activity in mouse splenocyte cultures (FIG. 58).

(Total IgG response)
According to the results of this study, the use of either CpG ODN 7909 or ODN 10106 significantly enhanced the antibody titer against HBsAg compared to the antigen alone (
p <0.0001), on the other hand Ag + CpG ODN 7909 or Ag + CpG OD
There was no significant difference in antibody titers in animals immunized with N 10106 (p =
0.86). Furthermore, control ODN did not significantly enhance the anti-HB response when used in combination with HBsAg (p = 0.86) (FIG. 59). The increase in total IgG levels is seen in CpG ODN 10606 when CpG ODN 7909 is used.
Slightly but significantly (p = 0.04) larger compared to when is used.

(The nature of the humoral response (ratio of IgG1 to IgG2a))
In mice, IgG isotype distribution is widely used as an indicator of the nature of the immune response, where a high IgG2a / IgG1 ratio indicates a Th1-biased immune response (Con
stant and Bottomory, 1997). In this study, the use of CpG ODN significantly enhanced IgG2a titers compared to when the antigen was used alone or in combination with the control ODN 2137 (Ag vs. 7909).
P <0.01 for Ag, p <0.001 for Ag vs. 10106, and Ag + 790
P <0.001 for 9 vs Ag + 2137, and Ag + 10106 vs Ag + 2137
P <0.01). However, the level of IgG2a response is CpG ODN 790
When either 9 or ODN 10106 is used in combination with HBsAg,
It was similar (p> 0.05). Thus, CpG ODN 7909 and ODN
Both 10106 are equally potent in their ability to induce a Th1-biased immune response as measured by increased levels of IgG2a over IgG1 (FIG. 60).

(Conclusion)
In vitro data using human peripheral mononuclear cells show that two molecules of the B class (7909 and 10106) behave very similar, if not identical, in the various assays performed. In some assays, ODN 10106 is ODN 7909.
Worked better than.

According to the results of the above mouse studies, CpG ODN 7909 and ODN 1010
6 has similar immune enhancing properties both in terms of its in vitro effect on the innate immune response and its ability to enhance antigen-specific responses in vivo when administered with an antigen.

(Equivalent)
The above written description is considered to be sufficient to enable one skilled in the art to practice the invention.
The present invention should not be limited in scope by the examples provided. This example is intended as a mere illustration of one aspect of the invention, and other functionally equivalent embodiments are within the scope of the invention. In addition to what is shown and described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims (1)

Invention described in the specification.

Images