Immunostimulatory nucleic acid molecules

Krieg , et al. April 28, 2

Patent Grant 7524828

U.S. patent number 7,524,828 [Application Number 10/921,086] was granted by the patent office on 2009-04-28 for immunostimulatory nucleic acid molecules. This patent grant is currently assigned to University of Iowa Research Foundation. Invention is credited to Joel N. Kline, Arthur M. Krieg.


United States Patent 7,524,828
Krieg ,   et al. April 28, 2009

Immunostimulatory nucleic acid molecules

Abstract

Nucleic acid sequences containing unmethylated CpG dinucleotides that modulate an immune response including stimulating a Th1 pattern of immune activation, cytokine production, NK lytic activity, and B cell proliferation are disclosed. The sequences are also useful a synthetic adjuvant.


Inventors: Krieg; Arthur M. (Wellesley, MA), Kline; Joel N. (Iowa City, IA)
Assignee: University of Iowa Research Foundation (Iowa City, IA)
Family ID: 42104324
Appl. No.: 10/921,086
Filed: August 18, 2004

Prior Publication Data

Document Identifier Publication Date
US 20050101557 A1 May 12, 2005

Related U.S. Patent Documents

Application Number Filing Date Patent Number Issue Date
09337584 Jun 21, 1999
08960774 Oct 30, 1997 6239116
08738652 Oct 30, 1996 6207646
08386063 Feb 7, 1995 6194388
08276358 Jul 15, 1994

Current U.S. Class: 514/44R; 424/278.1; 424/450; 424/457; 424/434; 424/409; 424/490; 424/184.1; 424/282.1
Current CPC Class: A61P 33/00 (20180101); A61P 31/04 (20180101); A61K 31/4706 (20130101); A61P 37/08 (20180101); A61K 31/7125 (20130101); A61P 11/06 (20180101); A61P 35/00 (20180101); A61K 39/39 (20130101); A61K 39/00 (20130101); A61P 37/04 (20180101); A61P 37/06 (20180101); A61P 43/00 (20180101); A61P 1/00 (20180101); A61P 17/06 (20180101); C07H 21/00 (20130101); A61P 31/12 (20180101); C12Q 1/68 (20130101); A61P 31/00 (20180101); A61P 19/02 (20180101); A61P 1/02 (20180101); C12N 15/117 (20130101); A61K 31/00 (20130101); C07H 21/04 (20130101); A61K 31/711 (20130101); A61P 1/04 (20180101); A61K 31/7048 (20130101); A61P 37/02 (20180101); C12N 2310/315 (20130101); C12N 2310/17 (20130101); A61K 2039/55561 (20130101)
Current International Class: A61K 39/00 (20060101); A61K 9/52 (20060101); A61K 9/50 (20060101); A61K 9/16 (20060101); A01N 43/04 (20060101); A61K 39/38 (20060101); A61K 9/127 (20060101); A61K 47/00 (20060101); A61F 13/00 (20060101); A61K 31/70 (20060101); A61K 45/00 (20060101); A01N 25/08 (20060101)
Field of Search: ;514/44 ;424/184.1,278.1,282.1,409,434,450,457,490

References Cited [Referenced By]

U.S. Patent Documents
3906092 September 1975 Hilleman et al.
5112605 May 1992 Jardieu
5248670 September 1993 Draper et al.
5498410 March 1996 Gleich
5585479 December 1996 Hoke et al.
5663153 September 1997 Hutcherson et al.
5679647 October 1997 Carson et al.
5681555 October 1997 Gleich
5723335 March 1998 Hutcherson et al.
5726160 March 1998 McMichael
5786189 July 1998 Locht et al.
5804566 September 1998 Carson et al.
5849719 December 1998 Carson et al.
5908620 June 1999 Tu et al.
5955059 September 1999 Gilchrest et al.
5955442 September 1999 McMichael
5994315 November 1999 Nyce et al.
6025339 February 2000 Nyce
6040296 March 2000 Nyce
6090791 July 2000 Sato et al.
6096721 August 2000 McMichael
6100244 August 2000 McMichael
6174872 January 2001 Carson et al.
6194388 February 2001 Krieg et al.
6207646 March 2001 Krieg et al.
6214806 April 2001 Krieg et al.
6218371 April 2001 Krieg et al.
6221882 April 2001 Macfarlane
6239116 May 2001 Krieg et al.
6339068 January 2002 Krieg et al.
6399630 June 2002 Macfarlane
6406705 June 2002 Davis et al.
6426336 July 2002 Carson et al.
6429199 August 2002 Krieg et al.
6479504 November 2002 Macfarlane et al.
6498148 December 2002 Raz
6514948 February 2003 Raz
6521637 February 2003 Macfarlane
6562798 May 2003 Schwartz
6589940 July 2003 Raz et al.
6610661 August 2003 Carson et al.
6653292 November 2003 Krieg et al.
6727230 April 2004 Hutcherson et al.
6797276 September 2004 Glenn et al.
6815429 November 2004 Agrawal
6821957 November 2004 Davis et al.
6843992 January 2005 Diamond
6887464 May 2005 Coleman et al.
6936261 August 2005 Granoff et al.
6943240 September 2005 Bauer et al.
6949520 September 2005 Hartmann et al.
6951845 October 2005 Carson et al.
7001890 February 2006 Wagner et al.
7208478 April 2007 Carson et al.
7223741 May 2007 Krieg
7271156 September 2007 Krieg et al.
2001/0044416 November 2001 McCluskie et al.
2002/0064515 May 2002 Krieg et al.
2002/0086839 July 2002 Raz et al.
2002/0091097 July 2002 Bratzler et al.
2002/0156033 October 2002 Bratzler et al.
2002/0164341 November 2002 Davis et al.
2002/0165178 November 2002 Schetter et al.
2002/0198165 December 2002 Bratzler et al.
2003/0022852 January 2003 Van Nest et al.
2003/0026782 February 2003 Krieg et al.
2003/0026801 February 2003 Weiner et al.
2003/0027782 February 2003 Carson et al.
2003/0049266 March 2003 Fearon et al.
2003/0050261 March 2003 Krieg et al.
2003/0050263 March 2003 Krieg et al.
2003/0050268 March 2003 Krieg et al.
2003/0055014 March 2003 Bratzler
2003/0059773 March 2003 Van Nest et al.
2003/0064064 April 2003 Dina et al.
2003/0078223 April 2003 Raz et al.
2003/0087848 May 2003 Bratzler et al.
2003/0091599 May 2003 Davis et al.
2003/0092663 May 2003 Raz et al.
2003/0100527 May 2003 Krieg et al.
2003/0104523 June 2003 Bauer et al.
2003/0109469 June 2003 Carson et al.
2003/0119773 June 2003 Raz et al.
2003/0125279 July 2003 Junghans et al.
2003/0133988 July 2003 Fearon et al.
2003/0139364 July 2003 Krieg et al.
2003/0148316 August 2003 Lipford et al.
2003/0148976 August 2003 Krieg et al.
2003/0166001 September 2003 Lipford
2003/0181406 September 2003 Schetter et al.
2003/0186921 October 2003 Carson et al.
2003/0191079 October 2003 Krieg et al.
2003/0203861 October 2003 Carson et al.
2003/0212026 November 2003 Krieg et al.
2003/0224010 December 2003 Davis et al.
2003/0232074 December 2003 Lipford et al.
2003/0232780 December 2003 Carson et al.
2003/0232856 December 2003 Macfarlane
2004/0006010 January 2004 Carson et al.
2004/0006034 January 2004 Raz et al.
2004/0009949 January 2004 Krieg
2004/0030118 February 2004 Wagner et al.
2004/0053880 March 2004 Krieg
2004/0067902 April 2004 Bratzler et al.
2004/0067905 April 2004 Krieg
2004/0087534 May 2004 Krieg et al.
2004/0087538 May 2004 Krieg et al.
2004/0092468 May 2004 Schwartz et al.
2004/0092472 May 2004 Krieg
2004/0106568 June 2004 Krieg et al.
2004/0131628 July 2004 Bratzler et al.
2004/0132685 July 2004 Krieg et al.
2004/0142469 July 2004 Krieg et al.
2004/0143112 July 2004 Krieg et al.
2004/0147468 July 2004 Krieg et al.
2004/0152649 August 2004 Krieg
2004/0152656 August 2004 Krieg et al.
2004/0152657 August 2004 Krieg et al.
2004/0162258 August 2004 Krieg et al.
2004/0162262 August 2004 Krieg et al.
2004/0167089 August 2004 Krieg et al.
2004/0171150 September 2004 Krieg et al.
2004/0171571 September 2004 Krieg et al.
2004/0181045 September 2004 Krieg et al.
2004/0198680 October 2004 Krieg
2004/0198688 October 2004 Krieg et al.
2004/0229835 November 2004 Krieg et al.
2004/0234512 November 2004 Wagner et al.
2004/0235770 November 2004 Davis et al.
2004/0235774 November 2004 Bratzler et al.
2004/0235777 November 2004 Wagner et al.
2004/0235778 November 2004 Wagner et al.
2004/0266719 December 2004 McCluskie et al.
2005/0004061 January 2005 Krieg et al.
2005/0004062 January 2005 Krieg et al.
2005/0009774 January 2005 Krieg et al.
2005/0032734 February 2005 Davis et al.
2005/0032736 February 2005 Krieg et al.
2005/0037403 February 2005 Krieg et al.
2005/0037985 February 2005 Krieg et al.
2005/0042203 February 2005 Davis et al.
2005/0043529 February 2005 Davis et al.
2005/0049215 March 2005 Krieg et al.
2005/0049216 March 2005 Krieg et al.
2005/0054601 March 2005 Wagner et al.
2005/0054602 March 2005 Krieg et al.
2005/0059619 March 2005 Krieg et al.
2005/0059625 March 2005 Krieg et al.
2005/0070491 March 2005 Krieg et al.
2005/0075302 April 2005 Hutcherson et al.
2005/0100983 May 2005 Bauer et al.
2005/0101554 May 2005 Krieg et al.
2005/0101557 May 2005 Krieg et al.
2005/0119273 June 2005 Lipford et al.
2005/0123523 June 2005 Krieg et al.
2005/0130911 June 2005 Uhlmann et al.
2005/0148537 July 2005 Krieg et al.
2005/0169888 August 2005 Hartman et al.
2005/0171047 August 2005 Krieg et al.
2005/0181422 August 2005 Bauer et al.
2005/0182017 August 2005 Krieg
2005/0197314 September 2005 Krieg et al.
2005/0215500 September 2005 Krieg et al.
2005/0215501 September 2005 Lipford et al.
2005/0233995 October 2005 Krieg et al.
2005/0233999 October 2005 Krieg et al.
2005/0239732 October 2005 Krieg et al.
2005/0239733 October 2005 Jurk et al.
2005/0239734 October 2005 Uhlmann et al.
2005/0239736 October 2005 Krieg et al.
2005/0244379 November 2005 Krieg et al.
2005/0244380 November 2005 Krieg et al.
2005/0245477 November 2005 Krieg et al.
2005/0250726 November 2005 Krieg et al.
2005/0256073 November 2005 Lipford et al.
2005/0267057 December 2005 Krieg
2005/0267064 December 2005 Krieg et al.
2005/0277604 December 2005 Krieg et al.
2005/0277609 December 2005 Krieg et al.
2006/0003955 January 2006 Krieg et al.
2006/0003962 January 2006 Ahluwalia et al.
2006/0019239 January 2006 Ivins et al.
2006/0019909 January 2006 Agrawal et al.
2006/0019916 January 2006 Krieg et al.
2006/0019923 January 2006 Davis et al.
2006/0058251 March 2006 Krieg et al.
2006/0089326 April 2006 Krieg et al.
2006/0094683 May 2006 Krieg et al.
2006/0140875 June 2006 Krieg et al.
2006/0154890 July 2006 Bratzler et al.
2006/0172966 August 2006 Lipford et al.
2006/0188913 August 2006 Krieg et al.
2006/0211639 September 2006 Bratzler et al.
2006/0211644 September 2006 Krieg et al.
2006/0229271 October 2006 Krieg et al.
2006/0241076 October 2006 Uhlmann et al.
2006/0246035 November 2006 Ahluwalia et al.
2006/0286070 December 2006 Hartmann et al.
2006/0287263 December 2006 Davis et al.
2007/0009482 January 2007 Krieg et al.
2007/0010470 January 2007 Krieg et al.
2007/0037767 February 2007 Bratzler et al.
2007/0065467 March 2007 Krieg et al.
2007/0066553 March 2007 Krieg et al.
2007/0066554 March 2007 Krieg et al.
2007/0078104 April 2007 Krieg et al.
2007/0129320 June 2007 Davis et al.
2007/0142315 June 2007 Forsbach et al.
2007/0184465 August 2007 Wagner et al.
2007/0202128 August 2007 Krieg et al.
2007/0224210 September 2007 Krieg et al.
2007/0232622 October 2007 Lipford et al.
2008/0009455 January 2008 Krieg et al.
2008/0026011 January 2008 Krieg et al.
2008/0031936 February 2008 Krieg et al.
2008/0045473 February 2008 Uhlmann et al.
Foreign Patent Documents
0 468 520 Jan 1992 EP
0 468 520 Jan 1992 EP
0 302 758 Mar 1994 EP
0 766 571 Apr 1997 EP
WO 91/12811 Sep 1991 WO
WO 92/03456 Mar 1992 WO
WO 92/18522 Oct 1992 WO
WO 92/21353 Dec 1992 WO
WO 94/19945 Sep 1994 WO
WO 95/05853 Mar 1995 WO
WO 95/26204 Oct 1995 WO
WO 96/02555 Feb 1996 WO
WO 96/32138 Oct 1996 WO
WO 96/35782 Nov 1996 WO
WO 96/40162 Dec 1996 WO
WO 97/28259 Aug 1997 WO
WO 98/14210 Apr 1998 WO
WO 98/16247 Apr 1998 WO
WO 98/18810 May 1998 WO
WO 98/37919 Sep 1998 WO
WO 98/40100 Sep 1998 WO
WO 98/49288 Nov 1998 WO
WO 98/52581 Nov 1998 WO
WO 98/55495 Dec 1998 WO
WO 99/11275 Mar 1999 WO
WO 99/52549 Oct 1999 WO
WO 99/56755 Nov 1999 WO
WO 99/58118 Nov 1999 WO
WO 99/62923 Dec 1999 WO
WO 00/06588 Feb 2000 WO
WO 00/14217 Mar 2000 WO
WO 00/16804 Mar 2000 WO
WO 00/20039 Apr 2000 WO
WO 00/54803 Sep 2000 WO
WO 00/62787 Oct 2000 WO
WO 00/67023 Nov 2000 WO
WO 01/02007 Jan 2001 WO
WO 01/12223 Feb 2001 WO
WO 01/45750 Jun 2001 WO
WO 01/68144 Sep 2001 WO
WO 03/000232 Jan 2003 WO
WO 03/094963 Nov 2003 WO
WO 03/101375 Dec 2003 WO
WO 2004/007743 Jan 2004 WO
WO 2004/026888 Apr 2004 WO
WO 2004/039829 May 2004 WO
WO 2004/094671 Nov 2004 WO
WO 2006/080946 Aug 2006 WO
WO 2007/031877 Mar 2007 WO
WO 2007/038720 Apr 2007 WO

Other References

Kitagaki et al., Clinical and Experimental Immunology, 2005, 143:249-259. cited by examiner .
Ferreira et al, Advances in Immunology, 2004, 84:79-129 abstract only. cited by examiner .
Racila et al, J. Allergy Clin. Immunol., 2005, 116:1202-1205. cited by examiner .
Weiss et al, Methods in Molecular Medicine, 2006, 127:253-267 abstract only. cited by examiner .
Macaubas et al, Current Drug Targets-Inflammation and Allergy, 2003, 2:175-186. cited by examiner .
Apielelberg et al, Current Opinion in Allergy and Clinical Immunology, 2002, 2/6:547-551 abstract only. cited by examiner .
Martin et al, Paediatric Respiratory Reviews, 2006, 7S:S106-S107. cited by examiner .
Klinman et al, J. Leukoocyte Biology, 2008, vol. 84, pp. 1-7. cited by examiner .
Iliev et al, Scand. J. Immunol., 2008, 67:370-376. cited by examiner .
Kline, Proc. Am. Thorac. Soc., 2007, 4:283-288. cited by examiner .
[No Author Listed] National Institute of Health, Publication No. 97-4051, Jul. 1997. cited by other .
Alm et al., Early BCG vaccination and development of atopy. Lancet. Aug. 9, 1997;350(9075):400-3. cited by other .
Agrawal et al., Antisense therapeutics: is it as simple as complementary base recognition? Mol Med Today. Feb. 2000;6(2):72-81. cited by other .
Agrawal et al., Novel immunomodulatory oligonucleotides prevent development of allergic airway inflammation and airway hyperresponsiveness in asthma. Int Immunopharmacol. Jan. 2004;4(1):127-38. cited by other .
Agrawal et al., Medicinal chemistry and therapeutic potential of CpG DNA. Trends Mol Med. Mar. 2002;8(3):114-21. cited by other .
Anitescu et al., Interleukin-10 functions in vitro and in vivo to inhibit bacterial DNA-induced secretion of interleukin-12. J Interferon Cytokine Res. Dec. 1997;17(12):781-8. cited by other .
Askenase et al., Gee whiz: CpG DNA allergy therapy! J Allergy Clin Immunol. Jul. 2000;106(1 Pt 1):37-40. cited by other .
Barnes et al., New treatments for asthma. European J Internal Medicine. 2000;11:9-20. cited by other .
Bauer et al., Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A. Jul. 31, 2001;98(16):9237-42. Epub Jul. 24, 2001. cited by other .
Bochner et al., Advances in mechanisms of allergy. J Allergy Clin Immunol. May 2004;113(5):868-75. cited by other .
Boushey et al., Targets for asthma therapy. Allerg Immunol (Paris) Nov. 2000;32(9):336-41. cited by other .
Campbell et al., Allergen immunotherapy: novel approaches in the management of allergic diseases and asthma. Clin Immunol. Dec. 2000;97(3):193-202. cited by other .
Chang et al., The effecet of CpG-oligodeoxynucleotides with different backbone structures and 3' hexameric deoxyriboguanosine run conjugation on the treatment of asthma in mice. J Allergy Clin Immunol. 2004;113(2):S323. Abstract 1196. cited by other .
Chiang et al., Ribavirin or CpG DNA sequence-modulated dendritic cells decrease the IgE level and airway inflammation. Am J Respir Crit Care Med. Sep. 1, 2003;168(5):575-80. cited by other .
Chisholm et al., Airway peptidoglycan and immunostimulatory DNA exposures have divergent effects on the development of airway allergen hypersensitivities. J Allergy Clin Immunol. Mar. 2004;113(3):448-54. cited by other .
Choudhury et al., In vivo role of p38 mitogen-activated protein kinase in mediating the anti-inflammatory effects of CpG oligodeoxynucleotide in murine asthma. J Immunol. Nov. 15, 2002;169(10):5955-61. cited by other .
Cockcroft et al., Comparative effects of inhaled salbutamol, sodium cromoglycate, and beclomethasone dipropionate on allergen-induced early asthmatic responses, late asthmatic responses, and increased bronchial responsiveness to histamine. J Allergy Clin Immunol. May 1987;79(5):734-40. cited by other .
Cooper et al., CPG 7909, an immunostimulatory TLR9 agonist oligodeoxynucleotide, as adjuvant to Engerix-B HBV vaccine in health adults: a double-blind phase I/II study. J Clin Immunol. Nov. 2004;24(6):693-701. cited by other .
Creticos et al., New approaches in immunotherapy: allergen vaccination with immunostimulatory DNA. Immunol. Allergy Clin North Am. Nov. 2004;24(4):569-81. cited by other .
Dziadzio et al., Handbook of Experimental Pharmacology, Pharmacology and Therapeutics of Asthma and COPD. 2004;161:273-85. cited by other .
Feleszko et al., Toll-like receptors--novel targets in allergic airway disease (probiotics, friends and relatives). Eur J Pharmacol. Mar. 2006;533(1-3):308-18. cited by other .
Erb et al., Infection of mice with Mycobacterium bovis-Bacillus Calmett-Guerin (BCG) suppressed allergen-induced airway eosinophilia. J Exp Med. Feb. 16, 1998;187(4):561-9. cited by other .
Fitch et al., The effect of an oral phosphodiesterase (PDE) 4 inhibitor bay 19-8004 in primate asthma models. American Thoracic Society. 2000. A52 Poster A94. cited by other .
Friedberg et al., Combination immunotherapy with a CpG oligonucleotide (1018 ISS) and rituximab in patients with non-Hodgkin lymphoma: increased interferon-alpha/beta-inducible gene expression, without significant toxicity. Blood. Jan. 15, 2005;105(2):489-95. Epub Sep. 9, 2004. cited by other .
Gibson et al., Cellular requirements for cytokine production in response to the immunomodulators imiquimod and S-27609. J Interferon Cytokine Res. Jun. 1995;15(6):537-45. cited by other .
Grunig, G. et al., Molecular regulation of Th2 immunity by dendritic cells. Pharmacol Ther. Apr. 2005;106(1):75-96. cited by other .
Gundel et al., Antigen-induced mediator release in primates. Am Rev Respir Dis. Jul. 1991;144(1):76-82. cited by other .
Gundel et al., Repeated antigen inhalation results in a prolonged airway eosinophilis and airway hyperresponsiveness in primates. J Appl Physiol. Feb. 1990;68(2):779-86. cited by other .
Hemmi et al., Toll-like receptor recognizes bacterial DNA. Nature. Dec. 7, 2000;408(6813):740-5. cited by other .
Hemmi et al., The roles of Toll-like receptor 9, MyD99, and DNA-dependent protein kinase catalytic subunit in the effects of two distinct CpG DNAs on dendritic cell subsets. J Immunol. Mar. 15, 2003;170(6):3059-64. cited by other .
Herz et al., BCG infection suppressed allergic sensitization and development of increased airway reactivity in an animal model. J Allergy Clin Immunol. Nov. 1998;102(5):867-74. cited by other .
Horner et al., Chapter 22: DNA-based immunotherapeutics for allergic disease. From: Microbial DNA and Host Immunity. 2002:279-87. cited by other .
Howarth et al., Influence of albuterol, cromolyn sodium and ipratropium bromide on the airway and circulating meditor responses to allergen bronchial provocation in asthma. Am Rev Respir Dis. Nov. 1985;132(5):986-92. cited by other .
Humlova et al., [Bacteria and their role in allergic diseases] Cas Lek Cesk. 2004;143(1):21-5. Review. Czech. Abstract Only. cited by other .
Hussain et al., CpG oligodeoxynucleotides: a novel therapeutic approach for atopic disorders. Curr Drug Targets Inflamm Allergy. Sep. 2003;2(3):199-205. cited by other .
Hussain et al., DNA, the immune system, and atopic disease. J Investig Dermatol Symp Proc. Jan. 2004;9(1):23-8. cited by other .
Infante-Duarte et al., Th1/Th2 balance in infection. Springer Semin Immunopathol. 1999;21(3):317-38. cited by other .
Jain et al., Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposire. Am J Physiol Lung Cell Mol Physiol. Nov. 2003;285(5):L1137-46. cited by other .
Jain et al., CpG DNA and immunotherapy of allergic airways diseases. Clin Exp Allergy. Oct. 2003;33(10):1330-5. cited by other .
Jain et al., The promise of CpG DNA in the treatment of asthma. Recent Res Develop Resp Crit Care Med. 2002;2:7-18. cited by other .
Jain et al., CpG-oligodeoxynucleotides inhibit airway remodeling in a murine model of chronic asthma. J Allergy Clin Immunol. Dec. 2001;110(6):867-72. cited by other .
Jain et al., CpG DNA: immunomodulation and remodelling of the asthmatic airway. Expert Opin Biol Ther. Sep. 2004;4(9):1533-40. cited by other .
Jones et al., Pharmacology of montelukast sodium (Singulair), a potent and selective leukotriene D4 receptor antagonist. Can J Physiol Pharmacol. Feb. 1995;73(2):191-201. cited by other .
Kitagaki et al., Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses. Clin Diagn Lab Immunol. Nov. 2002;9(6):1260-9. cited by other .
Kline et al., Effects of CpG DNA on Th1/Th2 balance in asthma. Curr Top Microbiol Immunol. 2000;247:211-25. cited by other .
Kline et al., DNA therapy for asthma. Curr Opin Allergy Clin Immunol. Feb. 2002;2(1):69-73. cited by other .
Krieg et al., Infection. In McGraw Hill Book. 1996: 242-3. cited by other .
Krieg et al., Lymphocyte activation by CpG dinucleotide motifs in prokaryotic DNA. Trends Microbiol. Feb. 1996;4(2):73-6. cited by other .
Kline et al., Induction of oral tolerance by CpG-ODNs in a murine model of asthma. J Allergy Clim Immunol. Feb. 2004;113(2):S254. Abstract 915. cited by other .
Krieg et al., CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. Apr. 6, 1995;374(6522):546-9. cited by other .
Krieg et al., Induction of systemic TH1-like innate immunity in normal volunteers following subcutaneous but not intravenous administration of CPG 7909, a synthetic B-class CpG oligodeoxynucleotide TLR9 agonist. J Immunother. Nov.-Dec. 2004;27(6):460-71. cited by other .
Krieg et al., Causing a commotion in the blood: immunotherapy progresses from bacteria to bacterial DNA. Immunol Today. Oct. 2000;21(10):521-6. cited by other .
Krown et al., Phase 1 trial with the interferon inducer polyI/poly-I-lysine (Poly ICL). Journal of Interferon Research. 1983; 3:281-90. cited by other .
Kuramoto et al., Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injection of a bacillus Calmette-Guerin nucleic acid fraction. Cancer Immunol Immunother. 1992;34(5):283-8. cited by other .
Lazarus et al., Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies. Genomics. Jan. 2003;81(1):85-91. cited by other .
Leigh et al., Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med. Nov. 1, 2002;166(9):1212-7. cited by other .
Litzinger et al., Fate of cationic liposomes and their complex with oligonucleotide in vivo. Biochim Biophys Acta. Jun. 11, 1996;1281(2):139-49. cited by other .
Liu et al., CpG ODN is an effective adjuvant in immunization with tumor antigen. J Invest Med. Sep. 7, 1997;45(7):333A. cited by other .
Liu et al., Hygiene hypothesis: fact or fiction? J Allergy Clin Immunol. Mar. 2003;111(3):471-8. cited by other .
Lukacs et al., Interleukin-4-dependent pulmonary eosinophil infiltration in a murine model of asthma. Am J Respir Cell Mol Biol. May 1994;10(5):526-32. cited by other .
Lukacs et al., C-C chemokine-induced eosinophil chemotaxis during allergic airway inflammation. J Leukoc Biol. Nov. 1996;60(5):573-8. cited by other .
McCluskie et al., The potential of oligodeoxynucleotides as mucosal and parenteral adjuvants. Vaccine. Mar. 21, 2001;19(17-19):2657-60. cited by other .
McCluskie et al., The potential of CpG oligodeoxynucleotides as mucosal adjuvants. Crit Rev Immunol. 2001;21(1-3):103-20. cited by other .
McCluskie et al., Route and method of delivery of DNA vaccine influence immune responses in mice and on-human primates. Mol Med. May 1999;5(5):287-300. cited by other .
Metzger et al., Oligonucleotide therapy of allergic asthma. J Allergy Clin Immunol. Aug. 1999;104(2 Pt 1):260-6. cited by other .
Milligan et al., Current concepts in antisense drug design. J Med Chem. Jul. 9, 1993;36(14):1923-37. cited by other .
Norman et al., Immunotherapy: 1999-2004. J Allergy Clin Immunol. Jun. 2004;113(6):1013-23. cited by other .
Padrid et la., CTLA4Ig inhibits airway eosinophilia and hyperresponsiveness by regulating the development of Th1/Th2 subsets in a murine model of asthma. Am J Respir Cell Mol Biol. Apr. 1998;18(4):453-62. cited by other .
Pare et al., Lung mechanics following antigen channelge of Ascaris suum-sensitive rhesus monkeys. J Appl Physiol. Nov. 1976;41(5 Pt. 1):668-76. cited by other .
Patterson et al., Inhibition of immunoglobulin E-mediated, antigen-induced money asthma and skin reactions by 5, 8, 11, 14-eicosatetraynoic acid. J Allergy Clin Immunol. Feb. 1981;67(2):146-52. cited by other .
Pisetsky et al., The immunologic properties of DNA. J Immunol. Jan. 15, 1996;156(2):421-3. cited by other .
Racila et al., Perspectives in asthma: molecular use of microbial products in asthma prevention and treatment. J Allergy Clin Immunol. Dec. 2005;116(6):1202-5. cited by other .
Redecke et al., Cutting edge: activation of Toll-like receptor 2 induces a Th2 immune response and promotes experimental asthma. J Immunol. Mar. 1, 2004;172(5):2739-43. cited by other .
Roy et al., Bacterial DNA in house and farm barn dust. J Allergy Clin Immunol. Sep. 2003;112(3):571-8. cited by other .
Sander et al., Sequential production of Th1 and Th2 cytokines in response to live bacillus Calmette-Guerin. Immunology. Dec. 1995;86(4):512-8. cited by other .
Satoh et al., The study of mechanisms in CpG oligodeoxynucleotides-induced aggravation in murine allergic contact dermititis to 2,4-dinitrofluorobenzene. Fukushima Igaku Zasshi. 2002;52(3):237-50. cited by other .
Serbrisky et al., CpG oligodeoxynucleotides can reverse Th2-associated allergic airway responses and alter the B7.1/B7.2 expression in a murine model of asthma. J Immunol. Nov. 15, 2000;165(10):5906-12. cited by other .
Siegrist et al., Co-administration of CpG oligonucleotides enhances the late affinity maturation process of human anti-hepatitis B vaccine response. Vaccine. Dec. 16, 2004;23(5):615-22. cited by other .
Silverman et al., BCG vaccination and atopy--unfinished business? Lancet. Aug. 9, 1997;350(9075):380-1. cited by other .
Silverman et al., Immunostimulatory DNA for asthma: better than eating dirt. Am J Respir Cell Mol Biol. Jun. 2003;28(6):645-7. cited by other .
Singh et al., Cationic microparticles are an effective delivery system for immune stimulatory CpG DNA. Pharm Res. Oct. 2001;18(10):1476-9. cited by other .
Sjolander et al., Iscoms containing purified Quillaja saponins upregulate both Th1-like and Th2-like immune responses. Cell Immunol. Apr. 10, 1997;177(1):69-76. cited by other .
Sonehara et al., Hexamer palindromic oligonucleotides with 5'-CD-3-' motif(s) induce production of interferon. J Interferon Cytokine Res. Oct. 1996;16(10):799-803. cited by other .
Sparwasser et al., Bacterial DNA causes septic shock. Nature. Mar. 27, 1997;386(6623):336-7. cited by other .
Stein et al., Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides. Antisense Res Dec. 1994 Summer;4(2):67-9. cited by other .
Stein et al., Non-antisense effects of oligodeoxynucleotides. Antisense Technology, 1997; ch11: 241-64. cited by other .
Stokes et al., Rationale for new treatments aimed at IgE immunomodulation. Ann Allergy Asthma Immunol. Sep. 2004;93(3):212-7. cited by other .
Tokunaga et al., A synthetic single-stranded DNA, poly(dG,dC), induces interferon-alpha/beta and -gamma, augments natural killer activity, and suppresses tumor growth. Jpn J Cancer Res. Jun. 1988;79(6):682-6. cited by other .
Tomai et al., Immunomodulating and antiviral activities of the imidazoquinoline S-28463. Antiviral Res. Nov. 1995;28(3):253-64. cited by other .
Turner et al., In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma. J Clin Invest. Jan. 15, 1996;97(2):381-7. cited by other .
Turner et al., Leukotriene D4 receptor antagonism reduces airway hyperresponsivess in monkeys. Pulm Pharmacol. Feb. 1994;7(1):49-58. cited by other .
Turner et al., Effects of rolipram on responses to acute and chronic antigen exposure in monkeys. Am J Respir Crit Care Med. May 1994;149(5):1153-9. cited by other .
Turner et al., Characterization of a primate model of asthma using anti-allergy/anti-asthma agents. Inflamm Res. May 1996;45(5):239-45. cited by other .
Vandenbulcke et al., The innate immune system and its role in allergic disorders. Int Arch Allergy Immunol. 2006;139(2):159-65. Abstract Only. cited by other .
Verthelyi et al., Immunoregulatory activity of CpG oligonucleotides in humans and nonhuman primates. Clin Immunol. Oct. 2003;109(1):64-71. cited by other .
Weiner et al., The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol. Oct. 2000;68(4):455-63. cited by other .
Witt et al., Phase I trial of an oral immunomodulator and interferon inducer in cancer patients. Cancer Res. Nov. 1, 1993;53(21):5176-80. cited by other .
Wong et al., Formoterol compared with beclomethasone and placebo on allergen-induced asthmatic responses. Am Rev Respir Dis. Nov. 1992;146(5 Pt 1):1156-60. cited by other .
Zhu et al., Modulation of ovalbumin-induced Th2 responses by second-generation immunomodulator oligonucleotides in mice. Int Immunopharmacol. Jul. 2004;4(7):851-62. cited by other .
Patent Interference No. 105,171. Decision of Motion under 37 CFR .sctn.41.125. Mar. 10, 2005. cited by other .
Patent Interference No. 105,171. Judgment and Order. Mar. 10, 2005. cited by other .
Patent Interference No. 105,171. Regents of the University of California Notice of Filing Appeal. Mar. 17, 2005. cited by other .
Patent Interference No. 105,171. Regents of the University of California. Brief of Appellant. Jul. 5, 2005. cited by other .
Patent Interference No. 105,171. University of Iowa and Coley Pharmaceutical Group, Inc. Brief of Appellees. Aug. 17, 2005. cited by other .
Patent Interference No. 105,171. Regents of the University of California. Reply Brief of Appellant. Sep. 6, 2005. cited by other .
Patent Interference No. 105,171. Regents of the University of California. Decision of CAFC. Jul. 17, 2006. cited by other .
Abed et al., Interferon-gama regulation of B lymphocyte differentiation: activation of B cells is a prerequisite for IFN-gamma-mediated inhibition of B cell differentiation. Cell Immunol. Feb. 1994;153(20:356-66. cited by other .
Adya N et al., Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 neat the conserved DNA-binding domain of CREB. Proc Natl Acad Sci USA 91(12):5642-6, Jun. 7, 1994. cited by other .
Agrawal et al., Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc Natl Acad Sci U S A. Sep. 1, 1991;88(17):7595-9. cited by other .
Anderson, G., et al., "TH2 and `TH2-like`cells in allergy and asthma: pharmacological perspectives", TiPS, 15:324-332, (1994). cited by other .
Angier, N., Microbe DNA Seen as Alien By Immune System, New York TImes, Apr. 11, 1995. cited by other .
Askew et al., "CpG DNA induces maturation of dendritic cells with distinct effects on nascent and recycling MHC-II antigen-processing mechanisms", Journal of Immunology, 165: 6889-95, 2000. cited by other .
Azad RF et al., Antiviral Activity of a Phosphorothioate Oligonucleotide Complementary to RNA of the Human Cytomegalovirus Major Immediate-Early Region. Antimicrobial Agents and Chemotherapy, 37:1945-1954, Sep. 1993. cited by other .
Azuma, Biochemical and Immunological Studies on Cellular Components o fTubercl Bacilli, Kekkaku, vol. 69, 9:45-55, 1992. cited by other .
Ballas ZK et al., Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol 157(5):1840-5, 1996. cited by other .
Bauer et al., DNA activates human immune cells through a CpG sequence-dependent manner. Immunology, Aug. 1999;97(4):699-705. cited by other .
Bayever, E., Systemic Administration of a Phosphorothioate Oligonucleotide with a Sequence Complementary to p53 for Acute Myelogenous leukemia and Myelodysplastic Syndrome: Initial Results of a Phase I Trial, Antisense Res. & Dev. (1993), 3:383-390. cited by other .
Bennett RM et al., DNA binding to human leukocytes. Evidence for a receptor-mediated association, internalization, and degradation of DNA. J Clin Invest 76(6):2182-90, 1985. cited by other .
Berg DJ et al., Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 96(5):2339-47, 1995. cited by other .
Blanchard DK et al., Interferon-gamma induction by lipopolysaccharide: dependence on interleukin 2 and macrophages. J Immunol 136(3):963-70, 1986. cited by other .
Blaxter et al., Genes expressed in Brugia malayi infective third stage larvae. Molecular and Biochemical Parasitology, 77:77-93. cited by other .
Boggs RT et al., Characterization and modulation of immune stimulation by modified oligonucleotides. Antisense Nucleic Acid Drug Dev 7(5):461-71, Oct. 1997. cited by other .
Bohle et al., Oligodeoxynucleotides containing CpG motifs induce IL-12, IL-18 and IFN-gamma production in cells from allergic individuals and inhibit IgE synthesis in vitro. Eur J Immunol. Jul. 1999;29(7):2344-53. cited by other .
Branda et al., Immune stimulation by an antisense oligomer complementary to the rev gene of HIV-1. Biochem Pharmacol. May 25, 1993;45(10):2037-43. cited by other .
Branda RF et al., Amplification of antibody production by phosphorothioate oligodeoxynucleotides. J. Lab Clin Med 128(3):329-38, Sep. 1996. cited by other .
Branda et al., Immune Stimulation by an Antisense Oligomer Complementary to the rev gene of HIV-1. Biochemical Pharmacology, vol. 45, 10:2037-2043, 1993. cited by other .
Brazolot-Millan et al., CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice. Proc Natl Acad Sci U S A. Dec. 22, 1998;95(26):15553-8. cited by other .
Briskin M et al., Lipopolysaccharide-unresponsive mutant pre-B-cell lines blocked in NF-kappa B activation. Mol Cell Biol 10(1):422-5, Jan. 1990. cited by other .
Broide et al., Modulation of asthmatic response by immunostimulatory DNA sequences. Springer Semin Immunopathol. 2000;221(1-2):117-24. Review. cited by other .
Broide et al., Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice. J Immunol. Dec. 15, 1998;161(12):7054-62. cited by other .
Broide et al., DNA-Based immunization for asthma. Int Arch Allergy Immunol. Feb.-Apr. 1999;118(2-4): 453-6. cited by other .
Broide, D. et al., "DNA-Based Immunization for Asthma", International Archives of Allergy and Immunology, 1999, pp. 453-456, vol. 118, S. Karger AG. Basel. cited by other .
Brunner et al., "Enhanced dendritic cell maturation by TNF-alpha or cytidine-phosphate-guanosine DNA drives T cell activation in vitro and therapeutic anti-tumor immune responses in vivo", Journal of Immunology, 165: 6278-6286, 2000. cited by other .
Capron et al., Immunologic aspects of schistosomiasis. Annu Rev Med. 1992;43:209-18. Review. Exhibit 2068. cited by other .
Carson et al., Oligonucleotide adjuvants for T Helper 1 (Th1)-specific vaccination. J Exp Med. Nov. 17, 1997;186(10):1621-2. Review. cited by other .
Chace et al., Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12. Clin Immunol Immunopathol. Aug. 1997;84(2):185-93. cited by other .
Chace, J. et al., Regulation of Differentiation in CD5+ and Conventional B Cells, Clinical Immunology and Immunopathology, (1993), 68:3:327-332. cited by other .
Chang YN et al., The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basel enhancers and cyclic AMP response elements. J Virol 64(10:264-77, Jan. 1990. cited by other .
Chu RS et al., CpG oligodeoxynucleotides act as adjuvants that switch on T helper 1 (Th1) immunity. J Exp Med 186(10):1623-31, Nov. 17, 1997. cited by other .
Cowdery JS et al., Bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides. J Immunol 156(12):4570-5, Jun. 15, 1996. cited by other .
Croft et al., Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J Exp Med. 1994 *C Nov. 1;180(5):1715-28. cited by other .
Crosby et al., The Early Responses Gene FGFI-C Encodes a Zinc Finger Transcriptional Activator and is a Member of the GCGGGGGCG (GSG) Element-Binding Protein Family. Mol. Cell. Biol., 2:3835-3841, 1991. cited by other .
Crystal, Transfer of Genes to Humans: Early Lessons and Obstacles to Success. Science, vol. 270, pp. 404-410, 1995. cited by other .
D'Andrea A et al., Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med 178(3):1041-8, 1993. cited by other .
Davis et al., "Use of CpG DNA for enhancing specific immune responses", Current Topics in Microbiology Immunology, 247: 171-83, 2000. cited by other .
Dunn et al., The three Es of cancer immunoediting. Annu Rev Immunol. 2004;22:329-60. Review. cited by other .
Durham et al., Immunotherapy and allergic inflammation. Clin Exp Allergy. Jan. 1991;21 Suppl 1:206-10. cited by other .
Englisch et al., Chemically Modified Oligonucleotides as Probes and Inhibitors, Angew. Chem. Int. Ed. Engl., 30:613-629, 1991. cited by other .
Erb KJ et al., Infection of mice with Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) suppressed allergen- induced airway eosinophilia. J Exp Med 187(4):561-9, Feb. 16, 1998. cited by other .
Etlinjer, Carrier sequence selection--one key to successful vaccines, Immunology Today, vol. 13, 2:52-55, 1992. cited by other .
Fox RI, Mechanism of action of hydroxychloroquine as an antirheumatic drug. Chemical Abstracts, 120:15, Abstract No. 182630 (Apr. 29, 1994). cited by other .
Francois et al., Examination of the inhibitory and stimulatory effects of IFN-alpha, -beta, and -gamma on human B-cell proliferation induced by various B-cell mitogens. Clin Immunol Immunopathol. Sep. 1988;48(3):297-306. cited by other .
Frissora et al., IFN-gamma-mediated inhibition of antigen receptor-induced B cell proliferation and CREB-1 binding activity required STAT-1 transcription factor. Eur J Immunol. Apr. 2003;33(4):907-12. cited by other .
Gura, T., Antisense Has Growing Pains. Science (1995), 270:575-576. cited by other .
Hadden J et al., Immunostimulants. TIPS, (1993), 141:169-174. cited by other .
Hadden J et al., Immunopharmacology, JAMA, (1992) 268:20:2964-2969. cited by other .
Halpern MD et al., Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol 167(1):72-8, 1996. cited by other .
Hartmann et al., "CpG DNA and LPS induce distinct patterns of activation in human monocytes", Gene Therapy, 6: 893-903, 1999. cited by other .
Hartmann et al., "Mechanism and function of a newly identified CpG DNA motif in human primary B cells", Journal of Immunology, 164: 944, 2000. cited by other .
Hartmann et al., "Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo", Journal of Immunology, 164: 1617, 2000. cited by other .
Hartmann et al., "CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells", Proceedings of the National Academy of Science USA, 96: 9305-9610, 1999. cited by other .
Hatzfeld J., Release of Early Human Hematopoietic Progenitors from Quiescence by Antisense Transforming Growth Factor .beta.1 or Rb Oligonucleotides, J. Exp. Med., (1991) 174:925-929. cited by other .
Heeg et al., CpG DNA as a Th1 trigger. Int Arch Allergy Immunol. Feb. 2000;121(2):87-97. Review. cited by other .
Highfield PE, Sepsis: the More, the Murkier. Biotechnology, 12:828, Aug. 12, 1994. cited by other .
Hoeffler JP et al., Identification of multiple nuclear factors that interact with cyclic adenosine 3',5'-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions. Mol Endocrinol 5(2):256-66, Feb. 1991. cited by other .
Hogg, J.C., "The pathology of asthma." Review article. APMIS 105: 735-745, 1997. cited by other .
Hopkin et al., BioMedNet, Issue 57, Jun. 25, 1999. cited by other .
Horner et al., Optimized conjugation ratios lead to allergen immunostimulatory oligodeoxynucleotide conjugates with retained immunogenicity and minimal anaphylactogenicity. J Allergy Clin Immunol. Sep. 2002;110(3):413-20. cited by other .
Horner et al., Immunostimulatory sequence oligodeoxynucleotide-based vaccination and immunomodulation: two unique but complementary strategies for the treatment of allergic diseases. J Allergy Clin Immunol. Nov. 2002;110(5):706-12. Review. cited by other .
Huang et al., Induction and regulation of Th1-inducing cytokines by bacterial DNA, *C lipopolysaccharide, and heat-inactivated bacteria. Infect Immun. Dec. 1999;67(12):6257-63. cited by other .
Iguchi-Ariga SM and Shaffner W, CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev 3(5):612-9, May 1989. cited by other .
Iho et al., "Oligodeoxynucleotides containing palindrome sequences with internal 5'-CpG-3' act directly on human NK and activated T cells to induce IFN-gamma production in vitro", Journal of Immunology, 163: 3642, 1999. cited by other .
Ikeda et al., Chapter 23: Immunostimulatory DNA for allergic asthma. microbial dna and Host Immunity. 2002. p. 289-299. cited by other .
Iverson, P., et al., "Pharmacokinetics of an Antisense Phosphorothioate Oligodeoxynucleotide against reve from Human Immunodeficiency Virus Type 1 in the Adule made Rate Following Single Injections and Continuous Infusion", Antisense Research and Development, (1994), 4:43-52. cited by other .
Ishikawa R et al., IFN induction and associated changes in splenic leukocyte distribution. J Immunol 150(9):3713-27, May 1, 1993. cited by other .
Jakob et al., "Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA", Journal of Immunology, 161(6): 3042-9, 1998. cited by other .
Jakob et al., "Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production: implications for the augmentation of Th1 responses", International Archives of Allergy Immunology, 118(2-4): 457-61, 1999. cited by other .
Jakway JP et al., Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide, and other bacterial products. J Immunol 137(7):2225-31, Oct. 1, 1986. cited by other .
Jaroszewski JW and Cohen JS, Cellular uptake of antisense oligonucleotides. Adv Drug Delivery Rev 6(3):235-50, 1991. cited by other .
Jilek et la., Antigen-independent suppression of the allergic immune response to bee venom phospholipase A(2) by DNA vaccination in CBA/J mice. J Immunol. Mar. 1, 2001;166(5):3612-21. cited by other .
Kataoka et al., "Antitumor activity of synthetic oligonucleotides with sequences from cDNA encoding proteins of Mycobacterium bovis BCG", Japan Journal of Cancer Research, 83: 244-247, 1992. cited by other .
Kataoka et la., "Immunotherapeutic potential in guinea-pig tumor model of deoxyribonucleic acid from Mycobacterium bovis BCG complexed with poly-L-lysine and carboxymethylcellulose", Japan Journal of Medical Science and Biology, 43(5):171-82, 1990. cited by other .
Kawano, K., et al., "Analysis and Regulation of interferon-gamma production by peripheral blood lymphocytes from patients with bronchial asthma", Abstract, Arerugi, 43:3:482-91, (1994). cited by other .
Kimura Y et al., Binding of Oligoguanylate to Scavenger Receptors Is Required for Oligonucleotides to Augment NK Cell Activity and Induce IFN, J. Biochem., vol. 116, 5:991-994, 1994. cited by other .
Kitagaki et al., Chapter 24: CpG oligodeoxynucleotides in asthma. Microbial DNA and Host Immunity. 2002. p. 301-314. cited by other .
Kline et al., CpG oligodeoxynucleotides do not require TH1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma. J Allergy Clin Immunol. Dec. 1999;104(6):1258-64. cited by other .
Kline et al., Treatment of established asthma in a murine model using CpG oligodeoxynucleotides. Am J Physiol Lung Cell Mol Physiol. Jul. 2002;283(1):L170-9. cited by other .
Kline et al., Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol. Mar. 15, 1998;160(6):2555-9. cited by other .
Kline JN et al., CpG motif oligonucleotides are effective in prevention of eosinophilic inflammation in a murine model of asthma. J Invest Med 44(7):380A, 1996. cited by other .
Kline JN et al., Immune redirection of CpG oligonucleotides. Conversion of a Th2 response to a Th1 response in a murine model of asthma. J Invest Med 45(3):282A, 1997. cited by other .
Kline JN et al., CpG oligonucleotides can reverse as well as prevent Th2-mediated inflammation in a murine model of asthma. J Invest Med 45(7):298A, 1997. cited by other .
Kline, J.N. et al., "CpG oligodeoxynucleotides do not require TH1 cytokines to prevent eosinophilic airway inflammation in a murine model of asthma", J. Allergy Clin. Immunol., Dec. 1999, pp. 1258-1264, vol. 104, No. 6. cited by other .
Klinman DM et al., CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci USA 93(7):2879-83, 1996. cited by other .
Klinman et al., "Immune recognition of foreign DNA: a cure for bioterrorism?", Immunity, 11: 123-129, 1999. cited by other .
Klinman et al., "Contribution of CpG motifs to the immunogenicity of DNA vaccines", Journal of Immunology, 158(8): 3635-9, 1997. cited by other .
Klinman et al., Immunotherpeutic applications of CpG-containing oligodeoxynucleotides. Drug News Perspect. Jun. 2000;13(5):289-96. cited by other .
Kohama et al., Immunostimulatory oligodeoxynucleotide induces TH1 immune response and inhibition of IgE antibody production to cedar pollen allergens in mice. J Allergy Clin Immunol. Dec. 1999;104(6):1231-8. cited by other .
Kovarik et al., "CpG oligodeoxynucleotides can circumvent the Th2 polarization of neonatal responses to vaccines but may fail to fully redirect Th2 responses established by neunatal priming", The Journal of Immunology, 162: 1611-1617, 1999. cited by other .
Kranzer et al. "CpG oligodeoxynucleotides can circumvent the Th2 polarization of neonatal responses to vaccines but may fail to fully redirect Th2 responses established by neonatal priming", Immunology, 99: 170, 2000. cited by other .
Krieg et al., "CpG motifs in bacterial DNA and their immune effects", Annual Reviews in Immunology, 20: 709, 2002. cited by other .
Krieg et al., Immune effects and therapeutic applications of CpG motifs in bacterial DNA. Immunopharmacology. Jul. 25, 2000;48(3):303-5. Review. cited by other .
Krieg et al., Abstract from 1996 meeting on Molecular Approaches to the Control of Infectious Diseases, Cold Spring Harbor Laboratory, Sep. 9-13, 1996. p. 116. cited by other .
Krieg et al., "Ehancing vaccines with immune stimulatory CpG DNA", Current Opinions Molecular Therapeutics, 3(1): 15-24, 2001. cited by other .
Krieg et al., A.M., "Immune effects and mechanisms of action of CpG motifs" Vaccine 19 (2001) 618-622. cited by other .
Krieg et al., Ernst Schering Researdh Found Workshop, (30): 105-18, 2001. cited by other .
Krieg et al., "Immune effects and mechanisms of action of CpG motifs", Vaccine, 19(6): 618-22, 2001. cited by other .
Krieg et al., "The role of CpG motifs in innate immunity", Current Opinions Immunology, 12: 35, 2000. cited by other .
Krieg et al., "Mechanism of Action in CpG DNA", Current Topics in Microbiology and Immunology, 247: 1-21, 2000. cited by other .
Krieg et al., "Mechanisms and therapeutic applications of immune stimulatory cpG DNA", Pharmacological Therapeutics, 84: 113, 1999. cited by other .
Krieg et al., "Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs", Proceedings of the National Academy of Science, 95: 12631-636, 1998. cited by other .
Krieg et al., "CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge", Journal of Immunology, 161(5): 2428-2434, 1998. cited by other .
Krieg et al., Chapter 17: Immune stimulation by oligonucleotides. p. 471-515, 2001. cited by other .
Krieg, The CpG motif: Implications for clinical immunology. BioDrugs. Nov. 1998; 10(5):341-6. cited by other .
Krieg, Mechanisms and applications of immune stimluatory CpG oligodeoxynucleotides. Biochim Biophys Acta. Dec. 10, 1999;1489(1):107-16 Review. cited by other .
Krieg et al., Signal transduction induced by immunostimulatory CpG DNA. Springer Semin Immunopathol. 2002;22(1-2):97-105. Review. cited by other .
Krieg et al., American College of Rheumatology 58th National Scientific Meeting. Minneapolis, Minnesota, Oct. 22, 1994. Abstracts. Arthritis Rheum. Sep. 1994;37(9 Suppl). cited by other .
Krieg, A.M. et al., "Immune effects and therapeutic applications of CpG motifs in bacterial DNA", Immunopharmacology, 2000, pp. 303-305, vol. 48, Elsevier Science B.V. cited by other .
Krieg AM, An innate immune defense mechanism based on the recognition of CpG motifs in microbial DNA. J Lab Clin Med. 128(2):128-33, 1996. cited by other .
Krieg AM et al., Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible. Antisense Res Dev 1(2):161-71, Summer 1991. cited by other .
Krieg AM et al., Oligodeoxynucleotide modifications determine the magnitude of B cell stimulation of CpG motifs. Antisense Nucleic Acid Drug Dev 6(2):133-9, Summer 1996. cited by other .
Krieg AM et al., "Modification of antisense phosphodiester oligodeoxynucleotides by a 5' cholesteryl moiety increases cellular association and improves efficacy", Proc. Natl. Acad. Sci., (1993), 90:1048-1052. cited by other .
Krieg AM et al., "CpG DNA: A Pathogenic Factor in Systemic Lupus Erythematosus?", Journal of Clinical Immunology, (1995) 15:6:284-292. cited by other .
Krieg AM et al, "Phosphorothioate Oligodeoxynucleotides: Antisense or Anti-Protein?", Antisense Research and Development, (1995), 5:241. cited by other .
Krieg AM et al., "Leukocyte Stimulation by Oligodeoxynucleotides", Applied Antisense Oligonucleotide Technology, (1998), 431-448. cited by other .
Krieg AM et al., CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374:546-9, 1995. cited by other .
Krieg AM et al, "The role of CpG dinuleotides in DNA vaccines", Trends in Microbiology, vol. 6, pp. 23-27, Jan. 1998. cited by other .
Krieg AM et al, A Role for Endogenous Retriviral Sequences in the Regulation of Lymphocyte Activation, the Journal of Immunology, vol. 143, 2448-2451, Oct. 15, 1989. cited by other .
Kuby et al., Editors, "Chapter 13: Cytokines", Immunology: Second Edition, 1994, W.H.Freeman and Company: New York, p. 297-322. cited by other .
Kuramoto et al., "Changes of host cell infiltration into Meth A fibrosarcoma tumor during the course of regression induced by injections of a BCG nucleic acid fraction", International Immunopharmacology, 14(5): 773-782, 1992. cited by other .
Kuramoto et al., "In situ infiltration of a natural killer-like cells induced by intradermal injection of the nucleic acid fraction from BCG", Microbiological Immunity, 33(11): 929-940, 1989. cited by other .
Kuramoto et al., Oligonucleotide Sequences Required for Natual Killer Cell Activation, Jpn. J. Cancer Res., 83:1128-1131, Nov. 1992. cited by other .
Leclerc et al., The preferential induction of a Th1 immune response by DNA-based immunization is mediated by the immunostimulatory effect of plasmid DNA. Cell Immunol. Aug. 1, 1997;179(2):97-106. cited by other .
Leibson et al., Role of gamma-interferon in antibody-producing responses. Nature. Jun. 28-Jul. 4, 1984;309(5971):799-801. cited by other .
Leonard et al., Interleukin-12: potential role in asthma theraph. BioDrugs. 2003;17(1):1-7. Review. cited by other .
Leonard et al., Conformation of Guanine 8-Oxoadenine Base Pairs in the Crystal Structure of d(CGCGAATT(08A)GCG). Biochemistry, 31(36):8415-8420, 1992. cited by other .
Lipford et al., "CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants", European Journal of Immunology, 27(9): 2340-4, 1997. cited by other .
Lipford et al., "Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines", European Journal of Immunology, 27: 3420-6, 1997. cited by other .
Lipford et al., Bacterial DNA as immune cell activator. Trends Microbiol. Dec. 1998;6(12):496-500. Review. cited by other .
Lotz et al., Effects of recombinant human interferons on rheumatoid arthritis B lymphocytes activated by Epstein-Barr virus. J Rheumatol. Feb. 1987;14(1):42-5. cited by other .
Ma et al., DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice. Vaccine. Sep. 10, 2002;20(27-28):3263-71. cited by other .
MacFarlane DE and Manzel L, Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds. J Immunol 160(3):1122-31, Feb. 1, 1998. cited by other .
Mardh et al., Alternaria alternata as caused of opportunistic fungal infections in man. Scand J Infect Dis Suppl. 1978;(16):36-40. cited by other .
Martin-Orozco et al., Enhancement of antigen-presenting cell surface molecules involved in cognate interactions by immunostimulatory DNA sequences. Int Immunol. Jul. 1999;11(7):1111-8. cited by other .
Mastrangelo et al. Seminars in Oncology. vol. 23, 1:4-21, 1996. cited by other .
Matson S and Krieg AM, Nonspecific suppression of [3H]thymidine incorporation by "control" oligonucleotides. Antisense Res Dev 2(4):325-30, Winter 1992. cited by other .
McCluskie et al., "CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice", Journal of Immunology, 161: 4463-6, 1998. cited by other .
McCluskie et al., CpG DNA as mucosal adjuvant. Vaccine, 18: 231-231, 2000. cited by other .
McCluskie et al., "Oral, intrarectal and intranasal immunizations using CpG and non-CpG oligodeoxynucelotides as adjuvants", Vaccine, 19: 413-422, 2001. cited by other .
McCluskie et al., "CpG DNA is an effective oral adjuvant to protein antigens in mice", *C Vaccine,19(7-8):950-7, 2000. cited by other .
McCluskie et al., "The role of CpG in DNA vaccines", Springer Seminars in Immunopathology, 22(1-2): 125-32, 2000. cited by other .
McCluskie et al., Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution. Mol Med. Oct. 2000;6(10):867-77. cited by other .
McIntyre KW et al., A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-kappa B p65 causes sequence-specific immune stimulation. Antisense Res Dev 3(4):309-22, Winter 1993. cited by other .
Messina et al., The Influence of DNA Structure on the in vitro Stimulation of Murine Lymphocytes by Natural and Synthetic Polynucleotide Antigens. Cellular Immunology, 147:148-157, 1993. cited by other .
Messina et al., Stimulation of in vitro Murine Lymphocyte Proliferation by Bacterial DNA. J. Immunol., vol. 147, 6:1759-1764, Sep. 15, 1991. cited by other .
Metzger et al., Enhancement of humoral immunity by interleukin-12. Ann N Y Acad Sci. Oct. 31, 1996;795:100-15. cited by other .
Mojcik, C., et al., "Administration of a Phosphorothioate Oligonucleotide Antisense Murine Endogenous Retroviral MCF env Causes Immune Effect in a Sequence-Specific Manner", Clinical Immunology and Immunopathology, (1993), 67:2:130-136. cited by other .
Mond et al., Recombinant interferon-gamma inhibits the B cell proliferative responses stimulated by soluble but not by Sepharose-bound anti-immunoglobulin antibody. J Immunol. Oct. 1985;135(4):2513-7. cited by other .
Mosmann et al., The expanding universe of T-cell substrates: Th1, Th2 and more. Immunol Today. Mar. 1996;17(3):138-46. Review. cited by other .
Mottram et al., A novel CDC2-related protein kinase from leishmania mexicana LmmCRK1 is post-transitionally regulated during the life cycle. J. Biol. Chem. 268:28, 21044-21052 (Oct. 1993). cited by other .
New England Biolabs 1988-1989 Catalog. cited by other .
Nyce JW and Metzger WJ, DNA antisense therapy for asthma in an animal model. Nature 385:721-725, Feb. 20, 1997. cited by other .
Park et al., The enhanced effect of a hexameric deoxyriboguanosine run conjugation to CpG oligodeoxynucleotides on protection against allergic asthma. J Allergy Clin Immunol. Oct. 2001;108(4):570-6. cited by other .
Parronchi et al., Phosphorothioate oligodeoxynucleotides promote the in vitro development of human allergen-specific CD4+ T cells into Th1 effects. J Immunol. Dec. 1, 1999;163(11):5946-53. cited by other .
Payette et al., "History of vaccines and positioning of current trends", Current Drugs Targets for Infection for Disorders, 1(3): 241-7, 2001. cited by other .
Pisetsky et al., "The influence of base sequence on the immunostimulatory properties of DNA", Immunity Research, 19: 35-46, 1999. cited by other .
Pisetsky et al., Immunological properties of bacterial DNA. Ann N Y Acad Sci. Nov. 27, 1995;772:152-63. Review. cited by other .
Pisetsky et al., Immune activation by bacterial DNA: a new genetic code. Immunity. Oct. 1996;5(4):303-10. Review. cited by other .
Pisetsky, D., "Stimulation of in vitro proliferation of murine lymphocytes by synthetic oligodeoxynucleotides", Molecular Biology Repairs, (1993) 18:217-221. cited by other .
Pisetsky et al., Stimulation of Murine Lymphocyte Proliferation by a Phosphorothioate Oligonucleotide with Antisense Activity for Herpes Simplex Virus. Life Science, vol. 54, pp. 101-107 (1994). cited by other .
Pisetsky, The Immunological Properties of DNA, The Journal of Immunology, pp. 421-423 (1996). cited by other .
Pisetsky, Immunological Consequences of Nucleic Acid Therapy, Antisense Research and Development, 5:219-225 (1995). cited by other .
Rankin et al., CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense Nucleic Acid Drug Dev. Oct. 2001;11(5):333-40. cited by other .
Raz et al., Intradermal gene immunization: the possible role of DNA uptake in the induction of cellular immunity to viruses. Proc Natl Acad Sci U S A. Sep. 27, 1994;91(20):9519-23. cited by other .
Raz E et al., Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. Proc Natl Acad Sci USA 93(10):5141-5, May 14, 1996. cited by other .
Raz et al., Potential role of immunostimulatory DNA sequences (ISS) in genetic immunization and autoimmunity. ACR Poster Session C: Cytokines and Inflammatory Mediators. Oct. 20, 1996; Abstract No. 615. cited by other .
Reynolds et al., Inhibition of B lymphocyte activation by interferon-gamma. J Immunol. Aug. 1, 1987;139(3):767-73. cited by other .
Ricci, M., et al., "T cells, cytokines, IgE and allergic airways inflammation", J Invest Allergol Clin Immunol, 4:5;214-220, (1994). cited by other .
Robinson, D.S. et al., "Predominant TH2-Like Bronchoalveolar T-Lymphocyte Population In Atopic Asthma" N Engl J Med 1992; 326:298-304. cited by other .
Roman M et al., Immunostimulatory DNA sequences function as T helper-1-promoting adjuvants. Nat Med 3(8):849-54, Aug. 1997. cited by other .
Sandrasagra et al., Discovery and development of respirable antisense therapeutics for asthma. Antisense Nucleic Acid Drug Dev. Jun. 2002;12(3):177-81. Review. cited by other .
Santeliz et al., Amb a 1-linked CpG oligodeoxynucleotides reverse established airway hyperresponsivenes in a murine model of asthma. J Allergy Clin Immunol. Mar. 2002;109(3):455-62. Exhibit 1041. cited by other .
Sato et al., Immunostimulatory DNA Sequences Necessary for Effective Intradermal Gene Immunization, Science, vol. 273, pp. 352-354, 1996. cited by other .
Schnell et al., Identification and characterization of a Saccharomyces cerevisiae gene (PAR1) conferring resistance to iron chelators. Eur. J. Biochem., 200:487-493, Sep. 1, 1991. cited by other .
Schwartz DA et al., Endotoxin responsiveness and grain dust-induced inflammation in the lower respiratory tract. Am J Physiol 267(5 Pt 1):L609-17, 1994. cited by other .
Schwartz DA et al., The role of endotoxin in grain dust-induced lung disease. Am J Respir Crit Care Med 152(2):603-8, 1995. cited by other .
Schwartz DA et al., CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract. J Clin Invest 100(1):68-73, Jul. 1, 1997. cited by other .
Schwartz et al., Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway. J Immunol. Jul. 1, 1999;163(1):224-31. cited by other .
Shirakawa T et al., The inverse association between tuberculin responses and atopic disorder. Science 275(5296):77-9, Jan. 3, 1997. cited by other .
Sidman et al., Gamma-interferon is one of several direct B cell-maturing lymphokines. Nature. Jun. 28-Jul. 4, 1984;309(5971):801-4. cited by other .
Spiegelberg, H., et al., "Recognition of T Cell Epitopes and Lymphokine Secretion by Rye Grass Allergen Lolium perenne I-Specific Human T Cell Clones", J of Immunology, 4706-4711, (1994). cited by other .
Sparwasser T et al., Marcophages sense pathogens via DNA motifs: induction of tumor necrosis factor-alpha-mediated shock. Eur J Immunol 27(7):1671-9, Jul. 1997. cited by other .
Speigelberg, H.L. et al., "DNA-based approaches to the treatment of allergies" Curr. Op. In Mol Therap. (2002) 4(1):64-71. cited by other .
Stein CA et al., Oligonucleotides as inhibitors of gene expression: a review. Cancer Research, 48:2659-2668, 1988. cited by other .
Stein et al., Antisense oligonucleotides as therapeutic agents--is the bulle treally magical? Science. Aug. 20, 1993;261(5124):1004-12. Review. cited by other .
Stuart, Marketplace strategies: The asthma challenge--Armed with a better understanding of the bad actors in the misguided immune response that causes allergies, companies hope to develop long-lasting treatments for asthma. Start-up. Apr. 1999; 12-20. cited by other .
Stull et al., Antigene, Ribozyme, and Aptamer Nucleic Acid Drugs: Progress and Prospects, Pharmaceutical Res., vol. 12, 4:465-483, 1995. cited by other .
Subramanian et al., Theoretical Considerations on the "Spine of Hydration" in the Minor Groose of d(CGCGAATTCGCG) d(GCGCTTAAGCGC): Monte Carlo Computer Simulation. Proc. Nat'l. Acad. Sci. USA, 85:1836-1840, 1988. cited by other .
Sun et al. "Type I interferon-mediated stimulation of T cells by CpG DNA", Journal of Experimental Medicine, 188: 2335, 1998. cited by other .
Sun et al. "Multiple effects of immunostimulatory DNA on T cells and the role of type I interferons", Springer Seminars in Immunopathology, 22: 77, 2000. cited by other .
Sur et al., Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J Immunol. May 15, 1999;162(10):6284-93. cited by other .
Tanaka T et al., An antisense Oligodeoxynucleotide complementary to a sequence in IG2b increases G2b germline transcripts stimulates B cell DNA synthesis and inhibits immunoglobulin secretion. J. Exp. Med., 175:597-607, 1992. cited by other .
Thorne PS., Experimental grain dust atmospheres generated by wet and dry aerosolization techniques. Am J Ind Med 25(1):109-12, 1994. cited by other .
Threadgill et al., "Mitogenic synthetic polynucleotides suppress the antibody response to a bacterial polysaccharide", Vaccine, 16(1): 76-82, 1998. cited by other .
Tighe et al., Conjugation of immunostimulatory DNA to the short ragweed allergen amb a 1 enhances its immunogenicity and reduces its allergenicity. J Allergy Clin Immunol. Jul. 2000;106(1 Pt 1):124-34. cited by other .
Tokunaga T et al., Synthetic Oligonucleotides with Particular Base Sequences form the cDNA Encoding Proteins of Myobacterium bovis BCG Induce Interferons and Activate Natural Killer Cells, Microbiol. Immunol., vol. 36, 1:55-66, 1992. cited by other .
Tokunga, Response of the organism to DNA--With a focus on immunostimulatory DNA. Kansen Ensho Meneki. 2001 Autumn; 31(3): 1-12. Japanese. Exhibits 1048 and 1049. cited by other .
Tokunaga et al., A Synthetic Single-Stranded DNA, Ply (dG, dC), Induces Interferon .alpha./.beta. and -.gamma., Augments Natural Killer Activity and Suppresses Tumor Growth, Jpn. J. Cancer Res., 79:682-686, Jun. 1988. cited by other .
Tournoy et al., Is Th1 the solution for Th2 in asthma? Clin Exp Allergy. Jan. 2002;32(1):17-29. Review. cited by other .
Uhlmann et al., Antisense Oligonucleotides: A New Therapeutic Principle. Chemical Reviews, 90:543-584, 1990. cited by other .
Van Uden et al., Immunostimulatory DNA and applications to allergic disease. J Allergy Clin Immunol. Nov. 1999;104(5):902-10. Review. cited by other .
Vlassov et al., Oligonucleotides in cells and in organisms: pharmacological considerations. in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS. 1991: 243-66. cited by other .
Wagner RW, Gene inhibition using antisense oligodeoxynucleotides. Nature, 372:L333-335, 1994. cited by other .
Wagner, Interactions between bacterial CpG-DNA and TLR9 bridge innate and adaptive immunity. Curr Opin Microbiol. Feb. 2002;5(1):62-9. Review. cited by other .
Walker, C., et al., "Activated T Cells and Cytokines in Bronchoalveolar Lavages from Patients with Various Lung Diseases Associated with Eosinophilia", Am J Respir Crit Care Med, 150:1038-1048, (1994). cited by other .
Wallace et al., Oligonucleotide probes for the screening of recombinant DNA libraries. Methods in Enzymology, 152:432-442 (1987). cited by other .
Wallner et al., Immunotherapy with T-cell-reactive peptides derived from allergens. Allergy. May 1994;49(5):302-8. Review. cited by other .
Weeratna et al., "CpG ODN can re-direct the Th bias of established Th2 immune responses in adult and yound mice", FEMS Immunology Medicine Microbiology, 32(1): 65-71, 2001. cited by other .
Weeratna et al., "CpG DNA induces stronger immune responses with less toxicity than other adjuvants", Vaccine, 18(17): 1755-62, 2000. cited by other .
Weeratna et al., Optimization strategies for DNA vaccines. Intervirology. 2000;43(4-6):218-26. cited by other .
Weeratna et al., CPG ODN allows lower dose of antigen against hepatitis B surface antigen in BALB/c mice. Immunol Cell Biol. Feb. 2003;81(1):59-62. cited by other .
Weiss R., Upping the Antisense Ante: Scientists bet on profits from reverse genetics. Science, 139:108-109, 1991. cited by other .
Wernette et al., "CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation", Veterinary Immunology and Immunopathology, 84(3-4): 223-236, 2002. cited by other .
Whalen et al., DNA-mediated immunization to the hepatitus B surface antigen. Activation and entrainment of the immune response. Ann N Y Acad Sci. Nov. 27, 1995;772:64-76. Review. cited by other .
Whalen R, DNA Vaccines for Emerging Infection Diseases: What If?, Emerging Infectiour Disease, vol. 2, 3:168-175, 1996. cited by other .
Wu GY et al., Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem., 263:14621-14624, 1988. cited by other .
Wu-Pong S., Oligonucleotides: Opportunities for Drug Therapy and Research. Pharmaceutical Technology, 18:102-114, 1994. cited by other .
Wyatt et al. "Combinatorially selected guanosine-quartet structure is a potent inhibitor of human immunodeficiency virue envelope-mediated cell fusion", Proceedings of the National Academy of Science USA, 91: 1356-60, 1994. cited by other .
Yamamoto, Cytokine production inducing action of oligo DNA. Rinsho Meneki. 1997;29(9): 1178-84. Japanese. Exhibits 1050, 1047, and 1046. cited by other .
Yamamoto S et al., DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol Immunol 36(9):983-97, 1992. cited by other .
Yamamoto S et al., In vitro augmentation of natural killer cell activity and production of interferon-alpha/beta and -gamma with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn J Cancer Res 79:866-73, Jul. 1988. cited by other .
Yamamoto S., Mode of Action of Oligonucleotide Fraction Extracted from Mycobacterium bovis BCG, Kekkaku, vol. 69, 9:29-32, 1994. cited by other .
Yamamoto S et al., Unique Palindromic Sequences in Synthetic Oligonucleotides are Required to Induce INF and Augment INF-Mediated Natural Killer Activity. J. Immunol., vol. 148, 12:4072-4076, Jun. 15, 1992. cited by other .
Yamamoto T et al., Ability of Oligonucleotides with Certain Palindromes to Induce Interferon Production and Augment Natural Killer Cell Activity is Associated with Their Base Length. Antisense Res. and Devel., 4:119-123, 1994. cited by other .
Yamamoto et al., Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity. Microbiol. Immunol., vol. 38, 10:831-836, 1994. cited by other .
Yamamoto T et al., Synthetic Oligonucleotides with Certain Palindromes Stimulate Interferon Production of Human Peripheral Blood Lymphocytes in vitro. Jpn. J. Cancer Res., 85:775-779, 1994. cited by other .
Yi, Ae-Kyung et al., IFN-.gamma. Promotes IL-6 and IgM Secretion in Response to CpG Motifs in Bacterial DNA and Oligonucleotides, The Journal of Immunology, pp. 558-564 (1996). cited by other .
Yi, Ae-Kying et al., Rapid Immune Activation by CpG Motifs in Bacterial DNA, The Journal of Immunology, pp. 5394-5402 (1996). cited by other .
Yi et al. "Rapid induction of mitogen-activated protein kinases by immune stimulatory CpG DNA", Journal of Immunology, 161: 4493, 1998. cited by other .
Yi et al. "CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry", Journal of Immunology, 160: 5898, 1998. cited by other .
Zhang et al., Antigen- and isotype-specific immune responses to a recombinant antigen-allergen chimeric (RAAC) protein. J Immunol. Jul. 15, 1993;151(2):791-9. cited by other .
Zhao et al., Pattern and kinetics of cytokine production following administration of phosphorothioate oligonucleotides in mice. Antisense Nucleic Acid Drug Dec. Oct. 1997;7(5):495-502. cited by other .
Zhao Q et al., Stage-specific oligonucleotide uptake in murine bone marrow B-cell precursors. Blood 84(11):3660-6, Dec. 1, 1994. cited by other .
Zhao Q et al., Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides. Antisense Res Dev 3(1):53-66, Spring 1993. cited by other .
Patent Interference No. 105,171. Iowa Opposition 1 (opposition to motion to designate additional claims as corresponding to the Count) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 2 (opposition to motion for judgment based on lack of written description support and introducing new matter) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 3 (opposition to motion for judgment based on anticipation) (Electronically filed, unsined). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 4 (opposition to motion for judgment based on obviousness) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 5 (opposition to motion for judgment based on anticipation) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 6 (opposition to motion for judgment based on inequitable conduct) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 7 (opposition to motion for benefit of an earlier application under 7 CFR 1.633(j)) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Opposition 8 (opposition to motion to add additional claims under 37 CFR 1.633 (2) and (i)) (Electronically filed, unsigned). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 3 (to Iowa Preliminary Motion 3 for judgment under 35 USC 135 (b)). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 4 (to Iowa Preliminary Motion 4 for judgment of no interference in fact). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 5 (to Iowa Preliminary Motion 5 for judgment that UC's claim is not enabled). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 6 (to Iowa Preliminary Motion 6 for judgment based on lack of adequate written description). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 7 (to Iowa Preliminary Motion 7 to redefine the interference). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 8 (to Iowa Preliminary Motion 8 to redefine the Count). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Response 9 (to Iowa Contingent Motion 9 for benefit). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Opposition 10 (to Iowa Contingent Motion 10 to redefine the interference). Sep. 9, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 3 (in support of Iowa Preliminary Motion 3 for judgment under 35 U.S.C. .sctn.135(b)) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 4 (in support of Iowa Preliminary Motion for judgment of no interference in fact) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 5 (in support of Iowa Preliminary Motion 5 for judgmetn that UC's claim 205 is not enabled) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 6 (in support of Iowa Preliminary Motion 6 for judgment based on lack of adequate written description) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 7 (in support of Iowa Preliminary Motion 7 to redefine the interference) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 8 (in support of Iowa Preliminary Motion 8 to redefine the count) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Reply 10 (in support of Iowa Preliminary Motion 10 to redefine the interference) (Electronically filed, unsigned). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents to the University of California Reply 1 (to Iowa's opposition to UC's motion to designate Iowa claims as corresponding to the Count). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 2 (to Iowa's opposition to UC Preliminary Motion 2 for Judgment). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Repy 3 (to Iowa's Opposition to UC Preliminary Motion 3 for Judgment). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 4 (to Iowa's Opposition to UC Preliminary Motion 4 for Judgment). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 5 (to Iowa's Opposition to UC Preliminary Motion 5 for Judgment). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 6 (to Iowa's opposition to UC Preliminary Motion 6 for judgment. Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 7 (to Iowa's Opposition to UC Preliminary Motion 7 for Benefit). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Reply 8 (to Iowa's Opposition to UC Preliminary Motion 8 to add additional claims). Oct. 15, 2004. cited by other .
Patent Interference No. 105,171. Miscellaneous Motion 1 (Unopposed Additional Request for Copies of File Histories). Apr. 15, 2004. cited by other .
Patent Interference No. 105,171. Miscellaneous Motion 2 (Unopposed Request for Addition to Iowa's File History). Apr. 15, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 3 (for Judgment Based on Failure to Comply with 35 U.S.C. 135(b)) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 4 (For Judgment of No. Interference in Fact) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 5 (For Judgment Based on Lack of Enablement) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 6 (For Judgment Based on Lack of an Adequate Written Description) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 7 (Motion to Redefine the Interference to Designate Claims as Not Corresponding to the Count) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 8 (Contingent Motion to Redefine Count) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 9 (Motion for Benefit of Earlier Application) (Electronically filed, unsigned). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Iowa Preliminary Motion 10 (Contingent Motion to Redefine the Interference By Adding A Continuation Application) (Electronically filed, unsigned). Jul. 2, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Statement. Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 1 (To Designate Additional Claims of the Iowa Patent as Corresponding to the Count). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California 2 (For Judgment Based on Lack of Written Description Support and Introducing New Matter). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 3 (For Judgment Based on Anticipation). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 4 (For Judgment Based on Obviousnes). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 5 (For Judgment Based on Anticipation). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 6 (For Judgment Based on Inequitable Conduct). Jun. 7, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 7 (For Benefit of an Earlier Application under 37 CFR 1.633(j)). Jul. 2, 2004. cited by other .
Patent Interference No. 105,171. Regents of the University of California Preliminary Motion 8 (To Add Additional Claims Under 37 CFR 1.633(c)(2) and (i)). Jul. 2, 2004. cited by other .
Amended Claims for U.S. Appl. No. 09/265,191, filed Mar. 10, 1999. cited by other .
[No Author Listed] Allergen Nomenclature, List of Allergens (as of Mar. 12, 2004). cited by other .
[No Author Listed] Expert panel report 2: Guidelines for the diagnosis and management of asthma--Clinical practice guidelines. National Institutes of Health Publication. Jul. 1997;No. 97-4051:iii-79. cited by other .
[No Author Listed] DNA is the primart genetic material. in Recombinant DNA, Second Edition. Waston et al., editors. Scientific American Books, New York. p. 24-5, 147. Exhibit 1003, 1992. cited by other .
[No Author Listed] Definitions. in The Dictionary of Immunology Fourth Edition. Herbert et al., editors. Academic Press, 1995. p. 10. Exhibit 1004. cited by other .
[No Author Listed] http://www.cancer.gov/newscenter/pressreleases/cancervaccines/print. Exhibit 1042, Mar. 27, 2003. cited by other .
[No Author Listed] http://www.cancer.gov/clinicaltrials/learning/cancervaccines/print. Exhibit 1043, Jun. 23, 2004. cited by other .
[No Author Listed] Recombinant DNA Technology. in Molecular Biology of the Cell. Alberts et al., editors. Garland Publishing, Inc. 1983. Exhibit 2027. cited by other .
[No Author Listed] Definitions. in Dorland's Illustrated Medical Dictionary, Twenty-sixth Edition. W.B. Saunders Company, 1981. Exhibit 2032. cited by other .
[No Author Listed] Macromolecules in Prokaryotic and Eukaryotic Cells. in Molecular Cell Biology, Darnell et al, Editors. W.H. Freeman and Company, 1986. Exhibit 2033. cited by other .
[No Author Listed] Definitions. in Immunology, Roitt et al., Editors. Gower Medical Publishing Company, 1985. Exhibit 2035. cited by other .
[No Author Listed] Definitions. in Webster's New Collegiate Dictionary. The G&C Merriam Company, 1981. p. 287. Exhibit 2070. cited by other .
Coley Pharmaceutical, Press Release, "Coley Pharmaceutical Group Updates Hepatitis C Drug Development Strategy," Jan. 22, 2007. cited by other .
Coley Pharmaceutical, Press Release, "Coley Pharmaceutical Group Announces Pfizer's Discontinuation of Clinical Trials for PF-3512676 Combined with Cytotoxic Chemotherapy in Advanced Non Small Cell Lung Cancer," Jun. 20, 2007. cited by other .
Kline et al., Effects of CpG DNA on Th1/Th2 balanse in asthma. Curr Top Microbiol Immunol. 2000;247:211-25. cited by other .
Kline et al., T-lymphocyte dysregulation in asthma. Proc Soc Exp Biol Med. Dec. 1994;207(3):243-53. cited by other .
Roman et al., Gene immunization for allergic disorders. Springer Semin Immunopathol. 1997;19(2):223-32. cited by other .
Patent Interference No. 105,526. Krieg Substantive Motion 1 (for unpatentability based on interference estoppel). (Electrinically filed, unsigned). cited by other .
Patent Interference No. 105,526.. Krieg Substantive Motion 2 (for judgment based on inadequate written description and/or enablement). (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526. Krieg Contingent Responsive Motion (to add new claims 104 and 105). (Electronically filed, unsigned). Jul. 25, 2007. cited by other .
Patent Interference No. 105,526. Krieg Substantive Motion 3 (for judgment based on prior art). (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526. Raz Motion 1 (Unpatentability of Krieg Claims under 35 U.S.C. .sctn. 112, First Paragraph). (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526. Raz Motion 2 (Raissing a Threshold Issue of No Interference-in-Fact). (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526.. Raz Motion 3 (Krieg's Claims are Unpatentable Over Prior Art Under 35 U.S.C. .sctn. 102(b)) (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526. Raz Motion 4 (To Designate Krieg Claims 46 and 82-84 as Corresponding to Count 1). (Electronically filed, unsigned). Jun. 18, 2007. cited by other .
Patent Interference No. 105,526. Raz Responsive Miscellaneous Motion 5 (To revive the Raz Parent Application) (Electronically filed, unsigned) Jul. 25, 2007. cited by other .
Patent Interference No. 105,526. Raz Contingent Responsive Motion 6 (To Add a New Claim 58) (Electronically filed, unsigned) Jul. 25, 2007. cited by other .
Patent Interference No. 105,526. Krieg Opposition 1 (Opposition to Motion for Lack of Enablement and Written Description) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Krieg Opposition 2 (to Raz Motion 2) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Krieg Opposition 3 (To Raz Motion 3) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Krieg Opposition 4 (Opposition to Motion for Designating Claims 46 and 82-84 as Corresponding to the Court) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Krieg Opposition 6 (Opposition to Raz Contingent Responsive Motion 6) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Raz Opposition 1 (Opposing Krieg Substantive Motion 1) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Raz Opposition 2 (Opposing Krieg Substantive Motion 2) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Raz Opposition 4 (Opposing Krieg Contingent Responsive Motion to Add New Claims 104 and 105) (Electronically filed, unsigned) Sep. 10, 2007. cited by other .
Patent Interference No. 105,526. Krieg Reply 1 (Reply to Raz opposition 1) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Krieg Reply 2 (Reply to Raz opposition 2) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Krieg Reply 4 (Reply to Raz opposition 4) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Raz Reply 1 (Reply to Krieg opposition 1) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Raz Reply 2 (Reply to Krieg opposition 2) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Raz Reply 3 (Reply to Krieg opposition 3) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Raz Reply 4 (Reply to Krieg opposition 4) Oct. 5, 2007. cited by other .
Patent Interference No. 105,526. Raz Reply 6 (Reply to Krieg opposition 6) Oct. 5, 2007. cited by other .
Arora et al., Immunomodulation by liposome entrapped allergen. Mol Cell Biochem. Sep. 21, 1990;97(2):173-9. Abstract Only. cited by other .
Gavett et al., Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice. J Exp Med. Nov. 1, 1995;182(5):1527-36. cited by other .
Goldberg et al., Beyond dager: unmethylated CpG dinucleotides and the immunopathogenesis of disease. Immunol Lett. Jul. 3, 2000;73(1):13-8. cited by other .
Kips et al., Interleukin-12 inhibits antigen-induced airways hyperresponsiveness in mice. Am J Respir Crit Care med. Feb. 1996;153(2):535-9. cited by other .
Krieg, Now I know my CpGs.Trends Microbiol. Jun. 2001;9(6):249-52. cited by other .
Mutwiri et al., Biological activity of immunostimulatory CpG DNA motifs in domestic animals Vet Immunol Immunopathol. Jan. 30, 2003;91(2):89-103. cited by other.

Primary Examiner: Minnifield; N. M
Attorney, Agent or Firm: Wolf, Greenfield & Sacks, P.C. Benson; Gregg C.

Government Interests



GOVERNMENT

The work resulting in this invention was supported in part by National Institute of Health Grant No. R29-AR42556-01. The U.S. Government may have rights in the invention.
Parent Case Text



RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 09/337,584, filed Jun. 21, 1999, currently pending, which is a divisional of U.S. Ser. No. 08/960,774, filed Oct. 30, 1997, now issued as U.S. Pat. No. 6,239,116B1 which is a continuation-in-part of U.S. Ser. No. 08/738,652, filed Oct. 30, 1996, now issued as U.S. Pat. No. 6,207,646B1 which is a continuation-in-part of U.S. patent application Ser. No. 08/386,063, filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388B1, which is a continuation-in-part of U.S. patent application Ser. No. 08/276,358, filed Jul. 15, 1994 which is now abandoned, each of which are incorporated herein by reference in their entirety.
Claims



We claim:

1. A method for treating an allergy in a subject, comprising administering to a subject an effective amount for desensitizing the subject against the occurrence of allergic reaction of a nucleic acid, having a sequence including at least the following formula: 5'X.sub.1X.sub.2CGX.sub.3X.sub.43' wherein C is unmethylated, wherein X.sub.1X.sub.2 and X.sub.3X.sub.4 are nucleotides, and wherein at least one internucleotide linkage has a phosphate modified backbone modification and wherein the nucleic acid has 8 to 40 nucleotides.

2. A method for treating an allergy in a subject, comprising orally administering to a subject an effective amount for desensitizing the subject against the occurrence of an allergic reaction of a immunostimulatory nucleic acid, having a sequence including at least the following formula: 5'X.sub.1X.sub.2CGX.sub.3X.sub.43' wherein C is unmethylated, and wherein X1X.sub.2 and X.sub.3X.sub.4 are nucleotides, and wherein at least one internucleotide linkage has a phosphorothioate modification and wherein the nucleic acid has 8 to 100 nucleotides.

3. A method for treating an allergy in a subject, comprising administering to a subject an effective amount for desensitizing the subject against the occurrence of an allergic reaction of a immunostimulatory nucleic acid, having a sequence including at least the following formula: 5'X.sub.1X.sub.2CGX.sub.3X.sub.43' wherein C is unmethylated, wherein X.sub.1X.sub.2 and X.sub.3X.sub.4 are nucleotides, and wherein the nucleic acid is administered by a route selected from the group consisting of transdermal and subcutaneous, and wherein at least one internucleotide linkage has a phosphorothioate modification and wherein the nucleic acid has 8 to 100 nucleotides.

4. A method for treating an allergy in a subject, comprising administering to a subject an effective amount for desensitizing the subject against the occurrence of an allergic reaction of a immunostimulatory nucleic acid, having a sequence including at least the following formula: 5'X.sub.1X.sub.2CGX.sub.3X.sub.43' wherein C is unmethylated, wherein X.sub.1X.sub.2 and X.sub.3X.sub.4 are nucleotides, and wherein the nucleic acid is administered in a formulation selected from the group consisting of a nucleic acid delivery complex, a liposome, a virosome, and a nanoparticle and wherein the nucleic acid has 8 to 100 nucleotides.
Description



FIELD OF THE INVENTION

The present invention relates generally to oligonucleotides and more specifically to oligonucleotides which have a sequence including at least one unmethylated CpG dinucleotide which are immunostimulatory.

BACKGROUND OF THE INVENTION

In the 1970s, several investigators reported the binding of high molecular weight DNA to cell membranes (Lerner, R. A., et al. 1971. "Membrane-associated DNA in the cytoplasm of diploid human lymphocytes." Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K., R. W. Wagner, P. K. McAllister, and B. Rosenberg. 1975. "Cell-surface-associated nucleic acid in tumorigenic cells made visible with platinum-pyrimidine complexes by electron microscopy." Proc. Natl. Acad. Sci. USA 72:928). In 1985, Bennett et al. presented the first evidence that DNA binding to lymphocytes is similar to a ligand receptor interaction: binding is saturable, competitive, and leads to DNA endocytosis and degradation into oligonucleotides (Bennett, R. M., G. T. Gabor, and M. M. Merritt, 1985. "J. Clin. Invest. 76:2182). Like DNA, oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable, sequence independent, and temperature and energy dependent fashion (reviewed in Jaroszewski, J. W. and J. S. Cohen. 1991. "Cellular uptake of antisense oligodeoxynlucleotides." Advanced Drug Deliver Reviews 6:235; Akhtar, S., Y. Shoji, and R. L. Juliano, 1992."Pharmaceutical aspects of the biological stability and membrane transport characteristics of antisense oligonucleotides." In: Gene Regulation: Biology of Antisense RNA and DNA, R. P. Erickson, and J. G. Izant, eds. Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschmidt, E. Fisher, C. J. Herrera, and A. M. Krieg. 1994. "Stage specific oligonucleotide uptake in murine bone marrow B cell precursors." Blood 84:3660). No receptor for DNA or ODN uptake has yet been cloned, and it is not yet clear whether ODN binding and cell uptake occurs through the same or a different mechanism from that of high molecular weight DNA.

Lymphocyte ODN uptake has been shown to be regulated by cell activation. Spleen cells stimulated with the B cell mitogen LPS had dramatically enhanced ODN uptake in the B cell population, while spleen cells treated with the T cell mitogen Con A showed enhanced ODN uptake by T but not B cells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L. A. Chrisey, and A. D. Steinberg. 1991. "Uptake of oligodeoxyribonucleotides by lymphoid cells is heterogeneous and inducible." Antisense Research and Development 1:161).

Several polynucleotides have been extensively evaluated as biological response modifiers. Perhaps the best example is poly (I,C) which is a potent inducer of IFN production as well as macrophage activator and inducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M. Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R. H. Wiltrout, and M. A. Chirigos. 1985. "Immunomodulatory effects in mice of polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose." Cancer Res. 45:1058; Wiltrout, R. H., R. R. Salup, T. A. Twilley, and J. E. Talmadge. 1985. "Immunomodulation of natural killer activity by polyribonucleotides." J. Biol. Respn. Mod. 4:512; Krown, S. E. 1986. "Interferons and interferon inducers in cancer treatment." Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp, J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W. G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P. Creekmore. 1992. "Polyinosinic-polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose in combination with interleukin-2 in patients with cancer: clinical and immunological effects." Canc. Res. 52:3005). It appears that this murine NK activation may be due solely to induction of IFN-.beta. secretion (Ishikawa, R., and C. A. Biron. 1993. "IFN inducation and associated changes in splenic leukocyte distribution". J. Immunol. 150:3713). This activation was specific for the robose sugar since deoxyribose was ineffective. Its potent in vitro antitumor activity led to several clinical trials using poly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (to reduce degradation by RNAse) Talmadge, J. E., et al., 1985. cited supra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986. cited supra); and Ewel, C. H., et al., 1992. cited supra). Unfortunately, toxic side effects have thus far prevented poly (I,C) from becoming a useful therapeutic agent.

Guanine ribonucleotides substituted at the C8 position with either a bromine or a thiol group are B cell mitogens and may replace "B cell differentiation factors" (Feldbush, T. L., and Z. K., Ballas. 1985. "Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation." J. Immunol. 134:3204; and Goodman, M. G. 1986. "Mechanism of synergy between T cell signals and C8-substituted guanine nucleosides in humoral immunity: B lymphotropic cytokines induce responsiveness to 8-mercaptoguanosine." J. Immunol. 136:3335). 8-mercaptoguanosine and 8-bromoguanosine also can substitute for the cytokine requirement for the generation of MHC restricted CTL (Feldbush, T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E. Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. "Activation of murine natural killer cells and macrophages by 8-bromoguanosine." J. Immunol. 140:3249), and synergize with IL-2 in inducing murine LAK generation (Thompson, R. A., and Z. K. Ballas. 1990. "Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as an IL-2-sparing agent in LAK generation." J. Immunol. 145:3524). The NK and LAK augmenting activities of these C8-substituted guanosines appear to be due to their induction of IFN (Thompson, R. A., et al. 1990. cited supra0. Recently, a 5' triphosphorylated thymidine produced by a mycobacterium was found to be mitogenic for a subset of human .gamma..delta. T cells (Constant, P., F. Davodeau, M.-A. Peyrat, Y. Poquet, G. Puzo, M. Bonneville, and J.-J. Fournie. 1994. "Stimulation of human .gamma..delta. T cells by nonpeptidic mycobacterial ligands." Science 264:267). This report indicated the possibility that the immune system may have evolved ways to preferentially respond to microbial nucleic acids.

Several observations suggest that certain DNA structures may also have the potential to activate lymphocytes. For example, Bell et al. reported that nucleosomal protein-DNA complexes (but not naked DNA) in spleen cell supernatants caused B cell proliferation and immunoglobulin secretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990. "Immunogenic DNA-related factors." J. Clin. Invest. 85:1487). In other cases, naked DNA has been reported to have immune effects. For example, Messina et al. have recently reported that 260 to 800 bp fragments of poly (dG)(dC) and poly (dGdC) were mitogenic for B cells (Messina, J. P., G. S. Gilkeson, and D. S. Piesetsky. 1993. "The influence of DNA structure on the in vitro stimulation of murine lymphocytes by natural and synthetic polynucleotide antigens." Cell. Immunol. 147:148). Tokunaga, et al. have reported that dGdC induces .gamma.-IFN and NK activity (Tokunaga, S. Yamamoto, and K. Nama. 1988. "A synthetic single-stranded DNA, poly(dG, dC), induces interferon-.alpha./b and -g, augments natural killer activity, and suppresses tumor growth." Jpn. J. Cancer Res. 79:682). Aside from such artificial homopolymer sequences, Pisetsky et al. reported that pure mammalian DNA has no detectable immune effects, but that DNA from certain bacteria induces B cell activation and immunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S. Pisetsky. 1991. "Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA." J. Immunol. 147:1759). Assuming that these data did not result from some unusual contaminant, these studies suggested that a particular structure or other characteristic of bacterial DNA renders it capable of triggering B cell activation. Investigations of mycobacterial DNA sequences have demonstrated that ODN which contain certain palindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto, T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. "Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF-mediated natural killer activity." J. Immunol. 148:4072; Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino, S. Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992. "Oligonucleotide sequences required for natural killer cell activation." Jpn. J. Cancer Res. 83:1128).

Several phosphorothioate modified ODN have been reported to induce in vitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E. Paul. 1992. "An antisense oligonucleotide complementary to a sequence in Ig2b increases g2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion." J. Exp. Med. 175:597; McIntyre, K. W., K. Lombard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D. Larigan, K. T. Landreth, and R. Narayanan. 1993. "A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-.kappa.B T65 causes sequence-specific immune stimulation." Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C. F. Reich. 1993. "Stimulation of murine lymphocyte proliferation by a phosphorothioate oligonucleotide with antisense activity for herpes simplex virus." Life Sciences 54:101). These reports do not suggest a common structural motif or sequence element in these ODN that might explain their effects.

The cAMP response element binding protein (CREB) and activating transcription factor (ATF) or CREB/ATF family of transcription factors is a ubiquitously expressed class of transcription factors of which 11 members have so far been cloned (reviewed on de Groot, R. P., and P. Sassone-Corsi: "Hormonal control of gene expression: Multiplicity and versatility of cyclic adenosine 3',5' -monophosphate-responsive nuclear regulators." Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson: "Transcriptional regulation by CREB and its relatives." Biochim. Biophys. Acta 1174:221, 1993). They all belong to the basic region/leucine zipper (bZip) class of proteins. All cells appear to express one or more CREB/ATF proteins, but the members expressed and the regulation of mRNA splicing appear to be tissue-specific. Differential splicing of activation domains can determine whether a particular CREB/ATF protein will be a transcriptional inhibitor or activator. Many CREB/ATF proteins activate viral transcription, but some splicing variants which lack the activation domain are inhibitory CREB/ATF proteins can bind DNA as homo- or hetero-dimers through the cAMP response element, the CRE, the consensus form of which is the unmethylated sequence TGACGTC (SEQ. ID. No. 103) (binding is abolished if the CpG is methylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: "CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA (SEQ. ID. No.104) abolishes specific factor binding as well as transcriptional activation." Genese & Develop. 3:612, 1989.

The transcriptional activity of the CRE is increased during B cell activation (Xie. H., T. C. Chiles, and T. L. Rothstein: "Induction of CREB activity via the surface Ig receptor of B cells." J. Immunol. 151:880, 1993). CREB/ATF proteins appear to regulate the expression of multiple genes through the CRE including immunologically important genes such as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R. Waterman, A. C. Webb, and P. E. Auron: "Transcription factors NF-IL6 and CREB recognize a common essential site in the human prointerleukin 1 gene." Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D. Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: "Antisense DNA inhibition of tumor growth induced by c-Ha-ras oncogene in nude mice." Cancer Res. 53:577, 1993), IFN-(Du, W., and T. Maniatis: "An ATF/CREB binding site protein is required for virus induction of the human interferon B gene." Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-1 (Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: "Binding of AP-1/CREB proteins and of MDBP to contiguous sites downstream of the human TGF-B1 gene." Biochim Biophys. Acta 1219:55, 1994), TGF-2, class II MHC (Cox, P. M., and C. R. Goding: "An ATF/CREB binding motif is required for aberrant constitutive expression of the MHC class II Dra promoter and activation by SV40 T-antigen." Nucl. Acids Res. 20:4881, 1992), E-selectin, GM-CSF, CD-8, the germline Ig constant region gene, the TCR V gene, and the proliferating cell nuclear antigen (Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M. B. Prystowsky: "Promoter activity of the proliferating-cell nuclear antigen gene is associated with inducible CRE-binding proteins in interleukin 2-stimulated T lymphocytes." Mol. Cell. Biol. 14:4233, 1994). In addition to activation through the cAMP pathway, CREB can also mediate transcriptional responses to changes in intracellular Ca.sup.++ concentration (Sheng, M., G. McFadden, and M. E. Greenberg: "Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB." Neuron 4:571, 1990).

The role of protein-protein interactions in transcriptional activation by CREB/ATF proteins appears to be extremely important. There are several published studies reporting direct or indirect interactions between NFKB proteins and CREB/ATF proteins (Whitley, et al., (1994) Mol. & Cell. Biol. 14:6464; Cogswell, et al., (1994) J. Immun. 153:712; Hines, et al., (1993) Oncogene 8:3189; and Du, et al., (1993) Cell 74:887. Activation of CREB through the cyclic AMP pathway requires protein kinase A (PKA), which phosphorylates CREB.sup.341 on ser.sup.133 and allows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J. R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G. Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: "Nuclear protein CBP is a coactivator for the transcription factor CREB." Nature 370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T. Smea, M. Karin, J. Feramisco, and M. Montminy: "Activation of cAMP and mitogen responsive genes relies on a common nuclear factor." Nature 370:226, 1994). CBP in turn interacts with the basal transcription factor TFIIB causing increased transcription. CREB also has been reported to interact with dTAFII 110, a TATA binding protein-:associated factor whose binding may regulate transcription (Ferreri, K., G. Gill, and M. Montminy: "The cAMP-regulated transcription factor CREB interacts with a component of the TFIID complex." Proc. Natl. Acad. Sci. USA 91:1210, 1994). In addition to these interactions, CREB/ATF proteins can specifically bind multiple other nuclear factors (Hoeffler, J. P., J. W. Lustbadfer, and C.-Y. Chen: "Identification of multiple nuclear factors that interact with cyclic adenosine 3',5'-monophosphate response element-binding protein and activating transcription factor-2 by protein-protein interactions." Mol. Endocrinol. 5:256, 1991) but the biologic significance of most of these interactions is unknown. CREB is normally thought to bind DNA either as a homodimer or as a heterodimer with several other proteins. Surprisingly, CREB monomers constitutively activate transcription (Krajewski, W., and K. A. W. Lee: "A monomeric derivative of the cellular transcription factor CREB functions as a constitutive activator." Mol. Cell. Biol. 14:7204, 1994).

Aside from their critical role in regulating cellular transcription, it has recently been shown that CREB/ATF proteins are subverted by some infectious viruses and retroviruses, which require them for viral replication. For example, the cytomegalovirus immediate early promoter, one of the strongest known mammalian promoters, contains eleven copies of the CRE which are essential for promoter function (Chang, Y.-N., S. Crawford, J. Stall, D. R. Rawlins, K.-T. Jeang, and G. S. Hayward: "The palindromic series I repeats in the simian cytomegalovirus major immediate-early promoter behave as both strong basal enhancers and cyclic AMP response elements." J. Virol. 64:264, 1990). At least some of the transcriptional activating effects of the adenovirus E1 A protein, which induces many promoters, are due to its binding to the DNA binding domain of the CREB/ATF protein, ATF-2, which mediates E1 A inducible transcription activation (Liu, F., and M. R. Green: "Promoter targeting by adenovirus E1A through interaction with different cellular DNA-binding domains." Nature 368:520, 1994). It has also been suggested that E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R. Sellers, D. M. Livingston, and R. Eckner: "E1A-associated p300 and CREB-associated CBP belong to a conserved family of coactivators." Cell 77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retrovirus which causes human T cell leukemia and tropical spastic paresis, also requires CREB/ATF proteins for replication. In this case, the retrovirus produces a protein, Tax, which binds to CREB/ATF proteins and redirects them from their normal cellular binding sites to different DNA sequences (flanked by; G- and G-rich sequences) present within the HTLV transcriptional enhancer (Paca-Uccaralertkun, S., L.-J. Zhao, N. Adya, J. V. Cross, B. R. Cullen, I. M. Boros, and C.-Z. Giam: "In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax." Mol. Cell. Biol. 14:456, 1994; Adya, N., L.-J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: "Expansion of CREB's DNA recognition specificity by Tax results from interaction with Ala-Ala-Arg at positions 282-284 near the conserved DNA-binding domain of CREB." Proc. Natl. Acad. Sci. USA 91:5642, 1994).

SUMMARY OF THE INVENTION

The present invention is based on the finding that certain nucleic acids containing unmethylated cytosine-guanine (CpG) dinucleotides activate lymphocytes in a subject and redirect a subject's immune response from a Th2 to a Th1 (e.g., by inducing monocytic cells and other cells to produce Th1 cytokines, including IL-12, IFN-.gamma. and GM-CSF). Based on this finding, the invention features, in one aspect, novel immunostimulatory nucleic acid compositions.

In one embodiment, the invention provides an isolated immunostimulatory nucleic acid sequence containing a CpG motif represented by the formula: 5'N.sub.1X.sub.1CGX.sub.2N.sub.23' wherein at least one nucleotide separates consecutive CpGs; X.sub.1 is adenine, guanine, or thymine; X.sub.2 is cytosine or thymine; N is any nucleotide and N.sub.1+N.sub.2 is from about 0-26 bases with the proviso that N.sub.1 and N.sub.2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.

In another embodiment, the invention provides an isolated immunostimulatory nucleic acid sequence contains a CpG motif represented by the formula: 5'N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.2C' wherein at least one nucleotide separates consecutive CpGs; X.sub.1X.sub.2 is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X.sub.3X.sub.4 is selected from the group consisting of TpT or CpT; N is any nucleotide and N.sub.1+N.sub.2 is from about 0-26 bases with the proviso that N.sub.1 and N.sub.2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.

In another embodiment, the invention provides a method of stimulating immune activation by administering the nucleic acid sequences of the invention to a subject, preferably a human. In a preferred embodiment, the immune activation effects predominantly a Th1 pattern of immune activation.

In another embodiment, the nucleic acid sequences of the invention stimulate cytokine production. In particular, cytokines such as IL-6, IL-12, IFN-.gamma., TNF-.alpha. and GM-CSF are produced via stimulation of the immune system using the nucleic acid sequences described herein. In another aspect, the nucleic acid sequences of the invention stimulate the lytic activity of natural killer cells (NK) and the proliferation of B cells.

In another embodiment, the nucleic acid sequences of the invention are useful as an artificial adjuvant for use during antibody generation in a mammal such as a mouse or a human.

In another embodiment, autoimmune disorders are treated by inhibiting a subject's response to CpG mediated leukocyte activation. The invention provides administration of inhibitors of endosomal acidification such as bafilomycin a, chloroquine, and monensin to ameliorate autoimmune disorders. In particular, systemic lupus erythematosus is treated in this manner.

The nucleic acid sequences of the invention can also be used to treat, prevent or ameliorate other disorders (e.g., a tumor or cancer or a viral, fungal, bacterial or parasitic infection). In addition, the nucleic acid sequences can be administered to stimulate a subject's response to a vaccine. Furthermore, by redirecting a subject's immune response from Th2 to Th1, the claimed nucleic acid sequences can be used to treat or prevent an asthmatic disorder. In addition, the claimed nucleic acid molecules can be administered to a subject in conjunction with a particular allergen as a type of desensitization therapy to treat or prevent the occurrence of an allergic reaction associated with an asthmatic disorder.

Further, the ability of the nucleic acid sequences of the invention described herein to induce leukemic cells to enter the cell cycle supports their use in treating leukemia by increasing the sensitivity of chronic leukemia cells followed by conventional ablative chemotherapy, or by combining the nucleic acid sequences with other immunotherapies.

Other features and advantages of the invention will become more apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C are graphs plotting dose-dependent IL-6 production in response to various DNA sequences in T cell depleted spleen cell cultures.

FIG. A 1. E. coli DNA (1) and calf thymus DNA (n) sequences and LPS (at 10.times. the concentration of E. coli and calf thymus DNA) (u).

FIG. 1B. Control phosphodiester oligodeoxynucleotide (ODN) 5' ATGGAAGGTCCAGTGTTCTC 3' (SEQ ID NO: 114) (n) and two phosphodiester CpG ODN 5' ATCGACCTACGTGCGTTCTC 3' (SEQ ID NO: 2) (u) and 5' TCCATAACGTTCCTGATGCT 3' (SEQ ID NO: 3) (1).

FIG. 1C. Control phosphorothioate ODN 5' GCTAGATGTTAGCGT 3' (SEQ ID NO: 4) (n) and two phosphorothioate CpG ODN 5' GAGAACGTCGACCTTCGAT 3' (SEQ ID NO: 5) (u) and 5' GCATGACGTTGAGCT 3' (SEQ ID NO: 6) (1). Data present the mean .+-.standard deviation of triplicates.

FIG. 2 is a graph plotting IL-6 production induced by CpG DNA in vivo as determined 1-8 hrs after injection. Data represent the mean from duplicate analyses of sera from two mice. BALB/c mice (two mice/group) were injected iv. with 100 .mu.l of PBS (o) of 200 .mu.g of CpG phosphorothioate ODN 5'TCCATGACGTTCCTGATGCT 3' (SEQ ID NO: 7) (n) or non-CpG phosphorothioate ODN 5' TCCATGAGCTTCCTGAGTCT 3' (SEQ ID NO: 8 (u).

FIG. 3 is an autoradiograph showing IL-6 mRNA expression as determined by reverse transcription polymerase chain reaction in liver, spleen, and thymus at various time periods after in vivo stimulation of BALB/c mice (two mice/group) injected iv with 100 .mu.l of PBS, 200 .mu.g of CpG phosphorothioate ODN 5' TCCATGACGTTCCTGATGCT 3' (SEQ ID NO: 7) or non-CpG phosphorothioate ODN 5' TCCATGAGCTTCCTGAGTCT 3' (SEQ ID NO: 8).

FIG. 4A is a graph plotting dose-dependent inhibition of CpG-induced IgM production by anti-IL-6. Splenic B-cells from DBA/2 mice were stimulated with CpG ODN 5' TCCAAGACGTTCCTGATGCT 3' (SEQ ID NO: 9) in the presence of the indicated concentrations of neutralizing anti-IL-6 (u) or isotype control Ab (1) and IgM levels in culture supernatants determined by ELISA. In the absence of CpG ODN, the anti-IL-6 Ab had no effect on IgM secretion (n).

FIG. 4B is a graph plotting the stimulation index of CpG-induced splenic B cells cultured with anti-L-6 and CpG S-ODN 5' TCCATGACGTTCCTGATGCT 3' (SEQ ID NO: 7) (u) or anti-IL-6-antibody only (n). Data present the mean .+-.standard deviation of triplicates.

FIG. 5 is a bar graph plotting chloramphenicol acetyltransferase (CAT) activity in WEH1-231 cells transfected with a promoter-less CAT construct (pCAT), positive control plasmid (RSV), or IL-6 promoter-CAT construct alone or cultured with CpG 5' TCCATGACGTTCCTGATGCT 3' (SEQ ID NO: 7) or non-CpG 5' TCCATGAGCTTCCTGAGTCT 3' (SEQ ID NO: 8) phosphorothioate ODN at the indicated concentrations. Data present the mean of triplicates.

FIG. 6 is a schematic overview of the immune effects of the immunostimulatory unmethylated CpG containing nucleic acids, which can directly activate both B cells and monocytic cells (including macrophages and dendritic cells) as shown. The immunostimulatory oligonucleotides do not directly activate purified NK cells, but render them competent to respond to IL-12 with a marked increase in their IFN-.gamma. secretion by NK cells, the immunostimulatory nucleic acids promote a Th1 type immune response. No direct activation of proliferation of cytokine secretion by highly purified T cells has been found. However, the induction of Th1 cytokine secretion by the immunostimulatory oligonucleotides promotes the development of a cytotoxic lymphocyte response.

FIG. 7 is an autoradiograph showing NF.kappa.B MRNA induction in monocytes treated with E. coli (EC) DNA (containing unmethylated CpG motifs), control (CT) DNA (containing no unmethylated CpG motifs) and lipopolysaccharide (LPS) at various measured times, 15 and 30 minutes after contact.

FIG. 8A shows the results from a flow cytometry study using mouse B cells with the dihydrorhodamine 123 dye to determine levels of reactive oxygen species. The dye only sample in Panel A of the figure shows the background level of cells positive for the dye at 28.6%. This level of reactive oxygen species was greatly increased to 80% in the cells treated for 20 minutes with PMA and ionomycin, a positive control (Panel B). The cells treated with the CpG oligo (TCCATGACGTTCCTGACGTT SEQ ID NO: 10) also showed an increase in the level of reactive oxygen species such that more than 50% of the cells became positive (Panel D). However, cells treated with an oligonucleotide with the identical sequence except that the CpGs were switched (TCCATGAGCTTCCTGAGTCT SEQ ID NO: 8) did not show this significant increase in the level of reactive oxygen species (Panel E).

FIG. 8B shows the results from a flow cytometry study using mouse B cells in the presence of chloroquine with the dihydrorhodamine 123 dye to determine levels of reactive oxygen species. Chloroquine slightly lowers the background level of reactive oxygen species in the cells such that the untreated cells in Panel A have only 4.3% that are positive. Chloroquine completely abolishes the induction of reactive oxygen species in the cells treated with CpG DNA (Panel B) but does not reduce the level of reactive oxygen species in the cells treated with PMA and ionomycin (Panel E).

FIG. 9 is a graph plotting lung lavage cell count over time. The graph shows that when the mice are initially injected with Schistosoma mansoni eggs "egg", which induces a Th2 immune response, and subsequently inhale Schistosoma mansoni egg antigen "SEA" (open circle), many inflammatory cells are present in the lungs. However, when the mice are initially given CpG oligo (SEQ ID NO: 10) along with egg, the inflammatory cells in the lung are not increased by subsequent inhalation of SEA (open triangles).

FIG. 10 is a graph plotting lung lavage eosinophil count over time. Again, the graph shows that when the mice are initially injected with egg and subsequently inhale SEA (open circle), many eosinophils are present in the lungs. However, when the mice are initially given CpG oligo (SEQ ID NO: 10) along with egg, the inflammatory cells in the lung are not increased by subsequent inhalation of the SEA (open triangles).

FIG. 11 is a bar graph plotting the effect on the percentage of macrophage, lymphocyte, neutrophil and eosinophil cells induced by exposure to saline alone; egg, then SEA; egg and SEQ ID NO: 11, then SEA; and egg and control oligo (SEQ ID NO: 11), then SEA. When the mice are treated with the control oligo at the time of the initial exposure to the egg, there is little effect on the subsequent influx of eosinophils into the lungs after inhalation of SEA. Thus, when mice inhale the eggs on days 14 or 21, they develop an acute inflammatory response in the lungs. However, giving a CpG oligo along with the eggs at the time of initial antigen exposure on days 0 and 7 almost completely abolishes the increase in eosinophils when the mice inhale the egg antigen on day 14.

FIG. 12 is a bar graph plotting eosinophil count in response to injection of various amounts of the protective oligo SEQ ID NO: 10.

FIG. 13 is a graph plotting interleukin 4 (IL-4) production (pg/ml) in mice over time in response to injection of egg, then SEA (open diamond); egg and SEQ ID NO: 10, then SEA (open circle); or saline, then saline (open square). The graph shows that the resultant inflammatory response correlates with the levels of the Th2 cytokine IL-4 in the lung.

FIG. 14 is a bar graph-plotting interleukin 12 (IL-12) production (pg/ml) in mice over time in response to injection of saline; egg, then SEA; or SEQ ID NO. 10 and egg, then SEA. The graph shows that administration of an oligonucleotide containing an unmethylated CpG motif can actually redirect the cytokine response of the lung to production of IL-12, indicating a Th1 type of immune response.

FIG. 15 is a bar graph plotting interferon gamma (IFN-.gamma.) production (pg/ml) in mice over time in response to injection of saline; egg, then saline; or SEQ ID NO: 10 and egg, then SEA. The graph shows that administration of an oligonucleotide containing an unmethylated CpG motif can also redirect the cytokine response of the lung to production of IFN-g, indicating a Th1 type of immune response.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the following terms and phrases shall have the meanings set forth below:

An "allergen" refers to a substance that can induce an allergic 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 (e.g., penicillin). Examples of natural, animal and plant allergens include proteins specific to the following genuses: Canine (Canis familiaris); Dermatophagoides (e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemuisfolia; Lolium (e.g., Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa); Betula (Betula verrucosa); Quercus (quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g., Plantago lanceolata); Parietaria (e.g., Parietaria officinalis or Parietaria judaica); Blattella (e.g., Blattella germanica); Apis (e.g., Apis multiflorum); Cupressus (e.g., Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g., Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g., Thuya orientalis), Chamaecyparis (e.g., Chamaecyparis obtusa); Periplaneta (e.g., Periplaneta americana); Agropyron (e.g., Agropyron repens); Secale (e.g., Secale cereale); Triticum (e.g., Triticum aestivum); Dactylis (e.g., Dactylis glomerata); Festuca (e.g., Festuca elatior); Poa (e.g., Poa pratensis or Poa compressa); Avena (e.g., Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g., Anthoxanthum odoratum); Arrhenatherum (e.g., Arrhenatherum elatius); Agrostis (e.g., Agrostis alba); Phleum (e.g., Phleum pratense); Phalaris (e.g., Phalaris arundinacea); Paspalum (e.g., Paspalum notatum); Sorghum (e.g., Sorghum halepensis) and Bromus (e.g., Bromus inermis).

An "allergy" refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include eczema, allergic rhinitis or coryza, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions.

"Asthma" refers to a disorder of the respiratory system characterized by inflammation, narrowing of the airways and increased reactivity of the airways to inhaled agents. Asthma is frequently, although not exclusively associated with atopic or allergic symptoms.

An "immune system deficiency" shall mean a disease or disorder in which the subject's immune system is not functioning in normal capacity or in which it would be useful to boost a subject's immune response for example to eliminate a tumor or cancer (e.g., tumors of the brain, lung (e.g., small cell and non-small cells), ovary, breast, prostate, colon, as well as other carcinomas and sarcomas) or an infection in a subject.

Examples of infectious virus include: Retroviridae (e.g., human irnmunodeficiency viruses, such as HIV-1, also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever virus); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herperviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola virsues, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta-hepatitides (thought-to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1--internally transmitted; class 2--parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. Intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter erogenes, Klebsiella pneuomiae, Pasturella multicoda, Bacteroides sp., Fusobacterium nucleatum, Sreptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomeyces israelli.

Examples of infectious fungi include: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans. Other infectious organisms (i.e., protists) include: Plasmodium falciparum and Toxoplasma gondii.

An "immunostimulatory nucleic acid molecule" refers to a nucleic acid molecule, which contains an unmethylated cytosine, guanine dinucleotide sequence (i.e., "CpG DNA" or DNA containing a cytosine followed by guanosine and linked by a phosphate bond) and stimulates (e.g., has a motogenic effect on, or induces or increases cytokine expression by) a vertebrate lymphocyte. An immunostimulatory nucleic acid molecule can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity.

In one preferred embodiment, the invention provides an isolated immunostimulatory nucleic acid sequence containing a CpG motif represented by the formula: 5'N.sub.1X.sub.1CGX.sub.2N.sub.23' wherein at least one nucleotide separates consecutive CpGs; X.sub.1 is adenine, guanine, or thymine; X.sub.2 is cytosine or thymine; N is any nucleotide and N.sub.1+N.sub.2 is from about 0-26 bases with the proviso that N.sub.1 and N.sub.2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.

In another embodiment the invention provides an isolated immunostimulatory nucleic acid sequence contains a CpG motif represented by the formula: 5'N.sub.1X.sub.1X.sub.2CGX.sub.3X.sub.4N.sub.23' wherein at least one nucleotide separates consecutive CpGs; X.sub.1X.sub.2 is selected from the group consisting of GpT, GpG, GpA, ApT and ApA; X.sub.3X.sub.4 is selected from the group consisting of TpT or CpT; N is any nucleotide and N.sub.1+N.sub.2 is from about 0-26 bases with the proviso that N.sub.1 and N.sub.2 do not contain a CCGG quadmer or more than one CCG or CGG trimer; and the nucleic acid sequence is from about 8-30 bases in length.

Preferably, the immunostimulatory nucleic acid sequences of the invention include X.sub.1X.sub.2 selected from the group consisting of GpT, GpG, GpA and ApA and X.sub.3X.sub.4 is selected from the group consisting of TpT, CpT and GpT (see for example, Table 5). For facilitating uptake into cells, CpG containing immunostimulatory nucleic acid molecules are preferably in the range of 8 to 30 bases in length. However, nucleic acids of any size (even many kb long) are immunostimulatory if sufficient immunostimulatory motifs are present, since such larger nucleic acids are degraded into oligonucleotides inside of cells. Preferred synthetic oligonucleotides do not include a CGG quadmer or more than one CCG or CGG trimer at or near the 5' and/or 3' terminals and/or the consensus mitdgenic CpG motif is not a palindrome. Prolonged immunostimulation can be obtained using stabilized oligonucleotides, where the oligonucleotide incorporates a phosphate backbone modification. For example, the modification is a phosphorothioate or phosphorodithioate modification. More particularly, the phosphate backbone modification occurs at the 5' end of the nucleic acid for example, at the first two nucleotides of the 5' end of the nucleic acid. Further, the phosphate backbone modification may occur at the 3' end of the nucleic acid for example, at the last five nucteotides of the 3' end of the nucleic acid.

Preferably the immunostimulatory CpG DNA is in the range of between 8 to 30 bases in size when it is an oligonucleotide. Alternatively, CpG dinucleotides can be produced on a large scale in plasmids, which after being administered to a subject are degraded into oligonucleotides. Preferred immunostimulatory nucleic acid molecules (e.g., for use in increasing the effectiveness of a vaccine or to treat an immune system deficiency by stimulating an antibody (i.e., humoral) response in a subject) have a relatively high stimulation index with regard to B cell, monocyte and/or natural killer cell responses (e.g., cytokine, proliferative, lytic or other responses).

The nucleic acid sequences of the invention stimulate cytokine production in a subject for example. Cytokines include but are not limited to IL-6, IL-12, IFN-.gamma., TNF-.alpha. and GM-CSF. Exemplary sequences include:

TABLE-US-00001 TCCATGTCGCTCCTGATGCT, (SEQ ID NO: 37) TCCATGTCGTTCCTGATGCT, (SEQ ID NO: 38) and TCGTCGTTTTGTCGTTTTGTCGTT. (SEQ ID NO: 46)

The nucleic acid sequences of the invention are also useful for stimulating natural killer cell (NK) lytic activity in a subject such as a human. Specific, but non-limiting examples of such sequences include:

TABLE-US-00002 TCGTCGTTGTCGTTGTCGTT, (SEQ ID NO: 47) TCGTCGTTTTGTCGTTTTGTCGTT, (SEQ ID NO: 46) TCGTCGTTGTCGTTTTGTCGTT, (SEQ ID NO: 49) GCGTGCGTTGTCGTTGTCGTT, (SEQ ID NO: 56) TGTCGTTTGTCGTTTGTCGTT, (SEQ ID NO: 48) TGTCGTTGTCGTTGTCGTT (SEQ ID NO: 50) and TCGTCGTCGTCGTT. (SEQ ID NO: 51)

The nucleic acid sequences of the invention are useful for stimulating B cell proliferation in a subject such as a human. Specific, but non-limiting examples of such sequences include:

TABLE-US-00003 TCCTGTCGTTCCTTGTCGTT, (SEQ ID NO: 52) TCCTGTCGTTTTTTGTCGTT, (SEQ ID NO: 53) TCGTCGCTGTCTGCCCTTCTT, (SEQ ID NO: 54) TCGTCGCTGTTGTCGTTTCTT, (SEQ ID NO: 55) TCGTCGTTTTGTCGTTTTGTCGTT, (SEQ ID NO: 46) TCGTCGTTGTCGTTTTGTCGTT (SEQ ID NO: 49) and TGTCGTTGTCGTTGTCGTT. (SEQ ID NO: 50)

In another aspect, the nucleic acid sequences of the invention are useful as an adjuvant for use during antibody production in a mammal. Specific, but non-limiting examples of such sequences include: TCCATGACGTTCCTGACGTT (SEQ ID NO: 10), GTCGTT (SEQ. ID. NO: 57), GTCGCT (SEQ. ID. NO. 58), TGTCGCT (SEQ. ID. NO: 101) and TGTCGTT (SEQ. ID. NO: 102). Furthermore, the claimed nucleic acid sequences can be administered to treat or prevent the symptoms of an asthmatic disorder by redirecting a subject's immune response from Th2 to Th1. An exemplary sequence includes

TABLE-US-00004 TCCATGACGTTCCTGACGTT. (SEQ ID NO: 10)

The stimulation index of a particular immunostimulatory CpG DNA can be tested in various immune cell assays. Preferably, the stimulation index of the immunostimulatory CpG DNA with regard to B-cell proliferation is at least about 5, preferably at least about 10, more preferably at least about 15 and most preferably at least about 20 as determined by incorporation of .sup.3H uridine in a murine B cell culture, which has been contacted with a 20 .mu.M of ODN for 20 h at 37.degree. C. and has peen pulsed with 1 .mu.Ci of .sup.3H uridine; and harvested and counted 4 h later as described in detail in Example 1. For use in vivo, for example to treat an immune system deficiency by stimulating a cell-mediated (local) immune response in a subject, it is important that the immunostimulatory CpG DNA be capable of effectively inducing cytokine secretion by monocytic cells and/or Natural Killer (NK) cell lytic activity.

Preferred immunostimulatory CpG nucleic acids should effect at least about 500 pg/ml of TNF-.alpha., 15 pg/ml IFN-.gamma., 70 pg/ml of GM-CSF 275 pg/ml of IL-6, 200 pg/ml IL-12, depending on the therapeutic indication, as determined by the assays described in Example 12. Other preferred immunostimulatory CpG DNAs should effect at least about 10%, more preferably at least about 15% and most preferably at least about 20% YAC-1 cell specific lysis or at least about 30, more preferably at least about 35 and most preferably at least about 40% 2C11 cell specific lysis as determined by the assay described in detail in Example 4.

A "nucleic acid" or "DNA" means multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the term refers to ribonucleotides as well as oligodeoxyribonucleotides. The term shall also include polynucleotides (i. e., a polynucleotide minus the phosphate) and any other organic base containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by oligonucleotide synthesis).

A "nucleic acid delivery complex" shall mean a nucleic acid molecule associated with (e.g., ionically or covalently bound to; or encapsulated within) a targeting means (e.g., a molecule that results in higher affinity binding to target cell (e.g., B-cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells). Examples of nucleic acid delivery complexes include nucleic acids associated with: a sterol (e.g., a ligand recognized by target cell specific receptor). Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex should be cleavable under appropriate conditions within the cell so that the nucleic acid is released in a functional form.

"Palindromic sequences" shall mean an inverted repeat (i.e:, a sequence such as ABCDEE'D'C'B'A' in which A and A' are bases capable of forming the usual Watson-Crick base pairs. In vivo, such sequences may form double stranded structures.

A "stabilized nucleic acid molecule" shall mean a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Unmethylated CpG containing nucleic acid molecules that are tens to hundreds of kbs long are relatively resistant to in vivo degradation. For shorter immunostimulatory nucleic acid molecules, secondary structure can stabilize and increase their effect. For example, if the 3' end of a nucleic acid molecule has self-complementarily to an upstream region, so that it can fold back and form a sort of stem loop structure, then the nucleic acid molecule becomes stabilized and therefore exhibits more activity.

Preferred stabilized nucleic acid molecules of the instant invention have a modified backbone. For use in immune stimulation, especially preferred stabilized nucleic acid molecules are phosphorothioate (i.e., at least one of the phosphate oxygens of the nucleic acid molecules is replaced by sulfur) or phosphorodithioate modified nucleic acid molecules. More particularly, the phosphate backbone modification occurs at the 5' end of the nucleic acid for example, at the first two nucleotides of the 5' end of the nucleic acid. Further, the phosphate backbone modification may occur at the 3' end of the nucleic acid for example, at the last five nucleotides of the 3' end of the nucleic acid. In addition to stabilizing nucleic acid molecules, as reported further herein, phosphorothioate-modified nucleic acid molecules (including phosphorodithioate-modified) can increase the extent of immune stimulation of the nucleic acid molecule, which contains an unmethylated CpG dinucleotide as shown herein. International Patent Application Publication Number WO 95/26204 entitled "Immune Stimulation By Phosphorothioate Oligonucleotide Analogs" also reports on the non-sequence specific immunostimulatory effect of phosphorothioate modified oligonucleotides. As reported herein, unmethylated CpG containing nucleic acid molecules having a phosphorothioate backbone have been found to preferentially activate B-cell activity, while unmethylated CpG containing nucleic acid molecules having a phosphodiester backbone have been found to preferentially activate monocytic (macrophages, dendritic cells and monocytes) and NK cells. Phosphorothioate CpG oligonucleotides with preferred human motifs are also strong activators of monocytic and NK cells.

Other stabilized nucleic acid molecules include: nonionic DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acid molecules which contain a diol, such as tetraethylenglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation.

A "subject" shall mean a human or vertebrate animal including a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey, rat, and mouse.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Preferred vectors are those capable of autonomous replication and expression of nucleic acids to which they are linked (e.g., an episome). Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

Certain Unmethylated CpG Containing Nucleic Acids Have B Cell Stimulatory Activity as Shown in vitro and in vivo

In the course of investigating the lymphocyte stimulatory effects of two antisense oligonucleotides specific for endogenous retroviral sequences, using protocols described in the attached Examples 1 and 2, it was surprisingly found that two out of twenty-four "controls" (including various scrambled, sense, and mismatch controls for a panel of "antisense" ODN) also mediated B cell activation and IgM secretion, while the other "controls" had no effect.

Two observations suggested that the mechanism of this B cell activation by the "control" ODN may not involve antisense effects 1) comparison of vertebrate DNA sequences listed in GenBank showed no greater homology than that seen with non-stimulatory ODN and 2) the two controls showed no hybridization to Northern blots with 10 .mu.g of spleen poly A+RNA. Resynthesis of these ODN on a different synthesizer or extensive purification by polyacrylamide gel electrophoresis or high pressure liquid chromatography gave identical stimulation, eliminating the possibility of an impurity. Similar stimulation was seen using B cells from C3H/HeJ mice, eliminating the possibility that lipopolysaccharide (LPS) contamination could account for the results.

The fact that two "control" ODN caused B cell activation similar to that of the two "antisense" ODN raised the possibility that all four ODN were stimulating B cells through some non-antisense mechanism involving a sequence motif that was absent in all of the other nonstimulatory control ODN. In comparing these sequences, it was discovered that all of the four stimulatory ODN contained CpG dinucleotides that were in a different sequence context from the nonstimulatory control.

To determine whether the CpG motif present in the stimulatory ODN was responsible for the observed stimulation, over 300 ODN ranging in length from 5 to 42 bases that contained methylated, unmethylated, or no CpG dinucleotides in various sequence contexts were synthesized. These ODNs, including the two original "controls" (ODN 1 and 2) and two originally synthesized as "antisense" (ODN 3D and 3M; Krieg, A. M. J. Immuno. 143:2448 (1989)), were then examined for in vitro effects on spleen cells (representative sequences are listed in Table 1). Several ODN that contained CpG dinucleotides induced B cell activation and IgM secretion; the magnitude of this stimulation typically could be increased by adding more CpG dinucleotides (Table 1; compare ODN 2 to 2a or 3D to 3Da and 3Db). Stimulation did not appear to result from an antisense mechanism or impurity. ODN caused no detectable proliferation of .gamma..delta. or other T cell populations.

Mitogenic ODN sequences uniformly became nonstimulatory if the CpG dinucleotide was mutated (Table 1; compare ODN 1 to 1a; 3D to 3Dc; 3M to 3Ma; and 4 to 4a) or if the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine (Table 1; ODN 1b, 2b, 3Dd, and 3Mb). Partial methylation of CpG motifs caused a partial loss of stimulatory effect (compare 2a to 2c, Table 1). In contrast, methylation of other cytosines did not reduce ODN activity (ODN 1c, 2d, 3De and 3Mc). These data confirmed that CpG motif is the essential element present in ODN that activate B cells.

In the course of these studies, it became clear that the bases flanking the CpG dinucleotide played an important role in determining the murine B cell activation induced by an ODN. The optimal stimulatory motif was determined to consist of a CpG flanked by two 5'. purines (preferably a GpA dinucleotide) and two 3'. pyrimidines (preferably a TpT or TpC dinucleotide). Mutations of ODN to bring the Cps motif closer to this ideal improved stimulation (e.g., Table 1, compare ODN 2 to 2e; 3M to 3Md) while mutations that disturbed the motif reduced stimulation (e.g., Table 1, compare ODN 3D to 3Df; 4 to 4b, 4c and 4d). On the other hand, mutations outside the CpG motif did not reduce stimulation (e.g., Table 1, compare ODN to 1d; 3D to 3Dg; 3M to 3Me). For activation of human cells, the best flanking bases are slightly different (See Table 5)).

Of those tested, ODNs shorter than 8 bases were non-stimulatory (e.g., Table 1, ODN 4e). Among the forty-eight 8 base ODN tested, a highly stimulatory sequence was identified as TCAACGTT (SEQ. ID. NO: 90) (ODN4) which contains the self complementary "palindrome" AACGTT (SEQ. ID. NO: 105). In further optimizing this motif, it was found that ODN containing Gs at both ends showed increased stimulation, particularly if the ODN were rendered nuclease resistant by phosphorothioate modification of the terminal internucleotide linkages. ODN 1585 (5' GGGGTCAACGTTGACGGGGG 3' (SEQ ID NO: 12)), in which the first two and last five internucleotide linkages are phosphorothioate modified caused an average 25.4 fold increase in mouse spleen cell proliferation compared to an average 3.2 fold increase in proliferation included by ODN 1638, which has the same sequence as ODN 1585 except that the 10 Gs at the two ends are replaced by 10 As. The effect of the G-rich ends is cis; addition of an ODN with poly G ends but no CpG motif to cells along with 1638 gave no increased proliferation. For nucleic acid molecules longer than 8 base pairs, non-palindromic motifs containing an unmethylated CpG were found to be more immunostimulatory.

TABLE-US-00005 TABLE 1 Stimulation Index' ODN Sequence (5' to 3').dagger. .sup.3H Uridine IgM Production 1 (SEQ ID NO:89) GCTAGACGTTAGCGT 6.1 .+-. 0.8 17.9 .+-. 3.6 1a (SEQ ID NO:4) ......T........ 1.2 .+-. 0.2 1.7 .+-. 0.5 1b (SEQ ID NO:13) ......Z........ 1.2 .+-. 0.1 1.8 .+-. 0.0 1c (SEQ ID NO:14) ............Z.. 10.3 .+-. 4.4 9.5 .+-. 1.8 1d (SEQ ID NO:6) ..AT......GAGC. 13.0 .+-. 2.3 18.3 .+-. 7.5 2 (SEQ ID NO:1) ATGGAAGGTCCAGCGTTCTC 2.9 .+-. 0.2 13.6 .+-. 2.0 2a (SEQ ID NO:15) ..C..CTC..G......... 7.7 .+-. 0.8 24.2 .+-. 3.2 2b (SEQ ID NO:16) ..Z..CTC.ZG..Z...... 1.6 .+-. 0.5 2.8 .+-. 2.2 2c (SEQ ID NO:17) ..Z..CTC..G......... 3.1 .+-. 0.6 7.3 .+-. 1.4 2d (SEQ ID NO:18) ..C..CTC..G......Z.. 7.4 .+-. 1.4 27.7 .+-. 5.4 2e (SEQ ID NO:19) ............A....... 5.6 .+-. 2.0 ND 3D (SEQ ID NO:20) GAGAACGCTGGACCTTCCAT 4.9 .+-. 0.5 19.9 .+-. 3.6 3Da (SEQ ID NO:21) .........C.......... 6.6 .+-. 1.5 33.9 .+-. 6.8 3Db (SEQ ID NO:22) .........C.......G.. 10.1 .+-. 2.8 25.4 .+-. 0.8 3Dc (SEQ ID NO:23) ...C.A.............. 1.0 .+-. 0.1 1.2 .+-. 0.5 3Dd (SEQ ID NO:24) .....Z.............. 1.2 .+-. 0.2 1.0 .+-. 0.4 3De (SEQ ID NO:25) .............Z...... 4.4 .+-. 1.2 18.8 .+-. 4.4 3Df (SEQ ID NO:26) .......A............ 1.6 .+-. 0.1 7.7 .+-. 0.4 3Dg (SEQ ID NO:27) .........CC.G.ACTG.. 6.1 .+-. 1.5 18.6 .+-. 1.5 3M (SEQ ID NO:28) TCCATGTCGGTCCTGATGCT 4.1 .+-. 0.2 23.2 .+-. 4.9 3Ma (SEQ ID NO:29) ......CT............ 0.9 .+-. 0.1 1.8 .+-. 0.5 3Mb (SEQ ID NO:30) .......Z............ 1.3 .+-. 0.3 1.5 .+-. 0.6 3Mc (SEQ ID NO:31) ...........Z........ 5.4 .+-. 1.5 8.5 .+-. 2.6 3Md (SEQ ID NO:7) ......A..T.......... 17.2 .+-. 9.4 ND 3Me (SEQ ID NO:32) ...............C..A. 3.6 .+-. 0.2 14.2 .+-. 5.2 4 (SEQ ID NO:90) TCAACGTT 6.1 .+-. 1.4 19.2 .+-. 5 4a (SEQ ID NO:91) ....GC.. 1.1 .+-. 0.2 1.5 .+-. 1.1 4b (SEQ ID NO:92) ...GCGC. 4.5 .+-. 0.2 9.6 .+-. 3.4 4c (SEQ ID NO:93) ...TCGA. 2.7 .+-. 1.0 ND 4d (SEQ ID NO:94) ..TT..AA 1.3 .+-. 0.2 ND 4e (Residue 2-8 of -....... 1.3 .+-. 0.2 1.1 .+-. 0.5 SEQ ID NO:90; SEQ ID 106) 4f (SEQ ID NO:95) C....... 3.9 .+-. 1.4 ND 4g (Residue 11-18 of --......CT 1.4 .+-. 0.3 ND SEQ ID NO:19; SEQ ID 117) 4h (SEQ ID NO:96) .......C 1.2 .+-. 0.2 ND Stimulation indexes are the means and std. dev. derived from at least 3 separate experiments, and are compared to wells cultured with no added ODN. ND = not done. CpG dinucleotides are underlined. Dots indicate identity; dashes indicate deletions. Z = 5 methyl cystosine.

TABLE-US-00006 TABLE 2 Identification of the optimal CpG motif for Murine IL-6 production and B cell activation. IL-6 (pg/ml).sup.a ODN SEQUENCE (5'-3') CH12.LX SPLENIC B CELL SI.sup.b IgM (ng/ml).sup.c 512 (SEQ ID No:28) TCCATGTCGGTCCTGATGCT 1300 .+-. 106 627 .+-. 43 5.8 .+-. 0.3 7315 .+-. 1324 1637 (SEQ ID No:33) ......C............. 136 .+-. 27 46 .+-. 6 1.7 .+-. 0.2 770 .+-. 72 1615 (SEQ ID No:34) ......G............. 1201 .+-. 155 850 .+-. 202 3.7 .+-. 0.3 3212 .+-. 617 1614 (SEQ ID No:35) ......A............. 1533 .+-. 321 1812 .+-. 103 10.8 .+-. 0.6 7558 .+-. 414 1636 (SEQ ID No:36) .........A.......... 1181 .+-. 76 947 .+-. 132 5.4 .+-. 0.4 3983 .+-. 485 1634 (SEQ ID No:37) .........C.......... 1049 .+-. 223 1671 .+-. 175 9.2 .+-. 0.9 6256 .+-. 261 1619 (SEQ ID No:38) .........T.......... 1555 .+-. 304 2908 .+-. 129 12.5 .+-. 1.0 8243 .+-. 698 1618 (SEQ ID No:7) ......A..T.......... 2109 .+-. 291 2596 .+-. 166 12.9 .+-. 0.7 10425 .+-. 674 1639 (SEQ ID No:3) .....AA..T.......... 1827 .+-. 83 2012 .+-. 132 11.5 .+-. 0.4 9489 .+-. 103 1707 (SEQ ID No:39) ......A..TC......... ND 1147 .+-. 175 4.0 .+-. 0.2 3534 .+-. 217 1708 (SEQ ID No:40) .....CA..TG......... ND 59 .+-. 3 1.5 .+-. 0.1 466 .+-. 109 Dots indicate identity; CpG dinucleotides are underlined; ND = not done .sup.aThe experiment was done at least three times with similar results. The level of IL-6 of unstimulated control cultures of both CH12.LX and splenic B cells was .ltoreq.10 pg/ml. The IgM level of unstimulated culture was 547 .+-. 82 ng/ml. CpG dinucleotides are underlined and dots indicate identity. .sup.b[.sup.3H] Uridine uptake was indicated as a fold increase (SI: stimulation index) from unstimulated control (2322.67 .+-. 213.68 cpm). Cells were stimulated with 20 .mu.M of various CpG O--ODN. Data present the mean .+-. SD of triplicates .sup.cMeasured by ELISA.

Other octamer ODN containing a 6 base palindrome with a TpC dinucleotide at the 5' end were also active (e.g., Table 1, ODN 4b, 4c). Other dinucleotides at the 5' end gave reduced stimulation (e.g., ODN 4f; all sixteen possible dinucleotides were tested). The presence of a 3' dinucleotide was insufficient to compensate for the lack of a 5' dinucleotide (e.g., Table 1, ODN 4g). Disruption of the palindrome eliminated stimulation in octamer ODN (e.g., Table 1, ODN 4h), but palindromes were not required in longer ODN.

The kinetics of lymphocyte activation were investigated using mouse spleen cells. When the cells were pulsed at the same time as ODN addition and harvested just four hours later, there was already a two-fold increase in .sup.3H uridine incorporation. Stimulation peaked at 12-48 hours and then decreased. After 24 hours, no intact ODN were detected, perhaps accounting for the subsequent fall in stimulation when purified B cells with or without anti-IgM (at a submitogenic dose) were cultured with CpG ODN, proliferation was found to synergistically increase about 10-fold by the two mitogens in combination after 48 hours. The magnitude of stimulation was concentration dependent and consistently exceeded that of LPS under optimal conditions for both. Oligonucteotides containing a nuclease resistant phosphorothioate backbone were approximately two hundred times more potent than unmodified oligonucleotides.

Cell cycle analysis was used to determine the proportion of B cells activated by CpG-ODN. CpG-ODN induced cycling in more than 95% of B cells. Splenic B lymphocytes sorted by flow cytometry into CD23- (marginal zone) and CD23+ (follicular) subpopulations were equally responsive to ODN-induced stimulation, as were both resting and activated populations of B cells isolated by fractionation over Percoll gradients. These studies demonstrated that CpG-ODN induced essentially all B cells to enter the cell cycle.

Immunostimulatory Nucleic Acid Molecules Block Murine B Cell Apostosis

Certain B cell lines, such as WEHI-231, are induced to undergo growth arrest and/or apoptosis in response to crosslinking of their antigen receptor by anti-IgM (Jakway, J. P. et al., "Growth regulation of the B lymphoma cell line WEHI-231 by anti-immunoglobulin, lipopolysaccharide and other bacterial products" J. Immunol. 137: 2225 (1986); Tsubata, T., J. Wu and T. Honjo: B-cell apoptosis induced yb antigen receptor crosslinking is blocked by a T-cell signal through CD40. " Nature 365: 645 (1993)). WEHI-231 cells are rescued from this growth arrest by certain stimuli such as LPS and by the CD40 ligand. ODN containing the CpG motif were also found to protect WEHI-231 from anti-IgM induced growth arrest, indicating that accessory cell populations are not required for the effect. Subsequent work indicates that CpG ODN induce Bcl-x and myc expression, which may account for the protection from apoptosis. Also, CpG nucleic acids have been found to block apoptosis in human cells. This inhibition of apoptosis is important, since it should enhance and prolong immune activation by CpG DNA.

Identification of the Optimal CpG Motif for Induction of Murine IL-6 and IgM Secretion and B Cell Proliferation.

To evaluate whether the optimal B cell stimulatory CpG motif was identical with the optimal CpG motif for IL-6 secretion, a panel of ODN in which the bases flanking the CpG dinucleotide were progressively substituted was studied. This ODN panel was analyzed for effects on B cell proliferation, Ig production, and IL-6 secretion, using both splenic B cells and CH12.LX cells. As shown in Table 2, the optimal stimulatory motif contains an unmethylated CpG flanked by two 5' purines and two 3' pyrimidines. Generally a mutation of either 5' purines to C were especially deleterious, but changes in 5' purines to T or 3' pyrimidines to purines had less marked effects. Based on analyses of these and scores of other ODN, it was determined that the optimal CpG motif for induction of IL-6 secretion is TGACGTT (SEQ. ID. NO: 108), which is identical with the optimal mitogenic and IgM-inducing CpG motif (Table 2). This motif was more stimulatory than any of the palindrome containing sequences studied (1639, 1707 and 1708).

Induction of Murine Cytokine Secretion by CpG motifs in Bacterial DNA or Oligonucleotides.

As described in Example 9, the amount of IL-6 secreted by spleen cells after CpG DNA stimulation was measured by ELISA. T cell depleted spleen cell cultures rather than whole spleen cells were used for in vitro studies following preliminary studies showing that T cells contribute little or nothing to the IL-6 produced by CpG DNA-stimulated spleen cells. As shown in Table 3, IL-6 production was markedly increased in cells cultured with E. coli DNA but not in cells cultured with calf thymus DNA. To confirm that the increased IL-6 production observed with E. coli DNA was not de to contamination by other bacterial products, the DNA was digested with DNAse prior to analysis. DNAse pretreatment abolished IL-6 production induced by E. coli DNA (Table 3). In addition, spleen cells from LPS-nonresponsive C2H/HeJ mouse produced similar levels of IL-6 in response to bacterial DNA. To analyze whether the IL-6 secretion induced by E. coli DNA was mediated by the unmethylated CpG dinucleotides in bacterial DNA, methylated E. coli DNA and a panel of synthetic ODN were examined. As shown in Table 3, CpG ODN significantly induced IL-6 secretion (ODN 5a, 5b, 5c) while CpG methylated E. coli DNA, or ODN containing methylated CpG (ODN 5f) or no CpG (ODN 5d) did not. Changes at sites other than CpG dinucleotides (ODN 5b) or methylation of other cytosines (ODN 5g) did not reduce the effect of CpG ODN. Methylation of a single CpG in an ODN with three CpGs resulted in a partial reduction in the stimulation (compare ODN 5c to 5e; Table 3).

TABLE-US-00007 TABLE 3 Induction of Murine IL-6 secretion by CpG motifs in bacterial DNA or oligonucleotides. Treatment IL-6 (pg/ml) calf thymus DNA .ltoreq.10 calf thymus DNA + DNase .ltoreq.10 E. coli DNA 1169.5 .+-. 94.1 E. coli DNA + DNase .ltoreq.10 CpG methylated E. coli DNA .ltoreq.10 LPS 280.1 .+-. 17.1 Media (no DNA) .ltoreq.10 ODN 5a SEQ. ID. No:115 ATGGACTCTCCAGCGTTCTC 1096.4 .+-. 372.0 5b SEQ. ID. No:19 .....AGG....A....... 1124.5 .+-. 126.2 5c SEQ. ID. No:15 ..C.......G......... 1783.0 .+-. 189.5 5d SEQ. ID. No:114 ....AGG..C..T...... .ltoreq.10 5e SEQ. ID. No:116 ..C.......G..Z...... 851.1 .+-. 114.4 5f SEQ. ID. No:16 ..Z......ZG..Z...... .ltoreq.10 5g SEQ. ID. No:18 ..C.......G......Z.. 1862.3 .+-. 87.26 T cell depleted spleen cells from DBA/2 mice were stimulated with phosphodiester modified oligonucleotides (O--ODN) (20 .mu.M), calf thymus DNA (50 .mu.g/ml) or E. coli DNA (50 .mu.g/ml) with or without enzyme treatment, or LPS (10 .mu.g/ml) for 24 hr. Data represent the mean (pg/ml) .+-. SD of triplicates. CpG dinucleotides are underlined and dots indicate identity. Z indicates 5-methylcytosine.

CpG Motifs Can Be Used as an Artificial Adjuvant.

Nonspecific simulators of the immune response are known as adjuvants. The use of adjuvants is essential to induce a strong antibody response to soluble antigens (Harlow and Lan, Antibodies: A Laboratory manual, Cold Spring harbor, N.Y. Current Edition; hereby incorporated by reference). The overall effect of adjuvants is dramatic and their importance cannot be overemphasized. The action of an adjuvant allows much smaller doses of antigen to be used and generates antibody responses that are more persistent. The nonspecific activation of the immune response often can spell the difference between success and failure in obtaining an immune response. Adjuvants should be used for first injections unless there is some very specific reason to avoid this. Most adjuvants incorporate two components. One component is designed to protect the antigen from rapid catabolism (e.g., liposomes or synthetic surfactants (Hunter et al. 1981)). Liposomes are only effective when the immunogen is incorporated into the outer lipid layer; entrapped molecules are not seen by the immune system. The other component is a substance that will stimulate the immune response nonspecifically. These substances act by raising the level of lymphokines. Lymphokines stimulate the activity of antigen-processing cells directly and cause a local inflammatory reaction at the site of injection. Early work relied entirely on heat-killed bacteria (Dienes 1936) or lipopolysaccharide (LPS) (Johnson et al. 1956). LPS is reasonably toxic, and, through analysis of its structural components, most of its properties as an adjuvant have been shown to be in a portion known as lipid A. Lipid A is available in a number of synthetic and natural forms that are much less toxic than LPS, but still retains most of the better adjuvant properties of parental LPS molecule. Lipid A compounds are often delivered using liposomes.

Recently an intense drive to find potent adjuvants with more acceptable side effects has led to the production of new synthetic adjuvants. The present invention provides the sequence 1826 TCCATGACGTTCCTGACGTT (SEQ ID NO: 10), which is an adjuvant including CpG containing nucleic acids. The sequence is a strong immune activating sequence and is a superb adjuvant, with efficacy comparable or superior to complete Freund's, but without apparent toxicity.

Titration of Induction of Murine IL-6 Secretion by CpG Motifs

Bacterial DNA and CpG ODN induced IL-6 production in T cell depleted murine spleen cells in a dose-dependent manner, but vertebrate DNA and non-CpG ODN did not (FIG. 1). IL-6 production plateaued at approximately 50 .mu.g/ml of bacterial DNA or 40 .mu.M of CpG O-ODN. The maximum levels of IL-6 induced by bacterial DNA and CpG ODN were 1-1.5 ng/ml and 2-4 ng/ml respectively. These levels were significantly greater than those seen-after stimulation by LPS (0.35 ng/ml) (FIG. 1A). To evaluate whether CpG ODN with a nuclease-resistant DNA backbone would also induce IL-6 production, S-ODN were added to T cell depleted murine spleen cells. CpG S-ODN also induced IL-6 production in a dose-dependent manner to approximately the same level as CpG O-ODN while non-CpG S-ODN failed to induce IL-6 (FIG. 1C). CpG S-ODN at a concentration of 0.05 .mu.M could induce maximal IL-6 production in these cells. This result indicated that the nuclease-resistant DNA backbone modification retains the sequence specific ability of CpG DNA to induce IL-6 secretion and that CpG S-ODN are more than 80-fold more potent than CpG O-ODN in this assay system.

Induction of Murine IL-6 Secretion by CpG DNA in vivo

To evaluate the ability of bacterial DNA and CpG S-ODN to induce IL-6 secretion in vivo, BALB/c mice were injected iv. with 100 .mu.g of E. coli DNA, calf thymus DNA, or CpG or non-stimulatory S-ODN and bled 2 hr after stimulation. The level of IL-6 in the sera from the E. coli DNA injected group was approximately 13 ng/ml while IL-6 was not detected in the sera from calf thymus DNA or PBS injected groups (Table 4). CpG S-ODN also induced IL-6 secretion in vivo. The IL-6 level in the sera from CpG S-ODN injected groups was approximately 20 ng/ml. In contrast, IL-6 was not detected in the sera from non-stimulatory S-ODN stimulated group (Table 4).

TABLE-US-00008 TABLE 4 Secretion of Murine IL-6 induced by CpG DNA stimulation in vivo. Stimulant IL-6 (pg/ml) PBS <50 E. coli DNA 13858 .+-. 3143 Calf Thymus DNA <50 CpG S-ODN 20715 .+-. 606 non-CpG S-ODN <50 Mice (2 mice/group) were i.v. injected with 100 .mu.l of PBS, 200 .mu.g of E. coli DNA or calf thymus DNA, or 500 .mu.g of CpG S-ODN or non-CpG control S-ODN. Mice were bled 2 hr after injection and 1:10 dilution of each serum was analyzed by IL-6 ELISA. Sensitivity limit of IL-6 ELISA was 5 pg/ml. Sequences of the CpG S-ODN is 5'GCATGACGTTGAGCT3' (SEQ. ID. No: 6) and of the non-stimulatory S-ODN is 5'GCTAGATGTTAGCGT3' (SEQ. ID. No: 4). Note that although there is a CpG in sequence 48, it is too close to the 3' end to effect stimulation, as explained herein. Data representation mean .+-. SD of duplicates. The experiment was done at least twice with similar results.

Kinetics of Murine IL-6 Secretion After Stimulation by CnG Motifs in vivo

To evaluate the kinetics of induction of IL-6 secretion by CpG DNA n vivo, BALB/c mice were injected iv. with CpG or control non-CpG S-ODN. Serum IL-6 levels were significantly increased within 1 hr and peaked at 2 hr to a level of approximately 9 ng/ml in the CpG S-ODN injected group (FIG. 2). Il-6 protein in sera rapidly decreased after 4 hr and returned to basal level by 12 hr after stimulation. In contrast to CpG DNA stimulated groups, no significant increase of IL-6 was observed in the sera from the non-stimulatory S-ODN or PBS injected groups (FIG. 2).

Tissue Distribution and Kinetics of IL-6. mRNA Expression Induced by CpG Motifs in vivo

As shown in FIG. 2, the level of serum IL-6 increased rapidly after CpG DNA stimulation. To investigate the possible tissue origin of this serum IL-6, and the kinetics of IL-6 gene expression in vivo after CpG DNA stimulation, BALB/c mice were injected iv with CpG or non-CpG S-ODN and RNA was extracted from liver, spleen, thymus, and bone marrow at various time points after stimulation. As shown in FIG. 3A, the level of IL-6 mRNA in liver, spleen, and thymus was increased within 30 min. after injection of CpG S-ODN. The liver IL-6 mRNA peaked at 2 hr post-injection and rapidly decreased and reached basal level 8 hr after stimulation (FIG. 3A). Splenic IL-6 mRNA peaked at 2 hr after stimulation and then gradually decreased (FIG. 3A). Thymus IL-6 mRNA peaked at 1 hr post-injection and then gradually decreased (FIG. 3A). IL-6 mRNA was significantly increased in bone marrow within 1 hr after CpG S-ODN injection but then returned to basal level. In response to CpG S-ODN, liver, spleen and thyrmus showed more substantial increases in IL-6 mRNA expression than the bone marrow.

Patterns of Murine Cytokine Expression Induced by CpG DNA

In vivo or in whole spleen cells, no significant increase in the protein levels of the following interleukins: IL-2, IL-3, IL-4, IL-5, or IL-10 was detected within the first six hours (Klinman, D. M. et al., (1996) Proc. Natl. Acad. Sci. USA 93:2879-2883). However, the level of TNF-.alpha. is increased within 30 minutes and the level of IL-6 increased strikingly within 2 hours in the serum of mice injected with CpG ODN. Increased expression of IL-12 and interferon gamma (IFN-.gamma.) mRNA by spleen cells was also detected within the first two hours.

TABLE-US-00009 TABLE 5 ODN Sequence (5'-3') IL-6.sub.1 TNF-.alpha..sub.1 IFN-.gamma..sub.1 GM-CSF IL-12 512 TCCATGTCGGTCCTGATGCT 500 140 15.6 70 250 SEQ ID NO:28 1637 ......C............. 550 16 7.8 15.6 16 SEQ ID NO:33 1615 ......G............. 600 145 7.8 45 145 SEQ ID NO:34 1614 ......A............. 550 31 0 50 31 SEQ ID N0:35 1636 .........A.......... 325 250 35 40 250 SEQ ID NO:36 1634 .........C.......... 300 400 40 85 400 SEQ ID NO:37 1619 .........T.......... 275 450 200 80 450 SEQ ID NO:38 1618 ......A..T.......... 300 60 15.6 15.6 62 SEQ ID NO:7 1639 .....AA..T.......... 625 220 15.6 40 220 SEQ ID NO:3 1707 ......A..TC......... 300 70 17 0 70 SEQ ID NO:39 1708 .....CA..TG......... 270 10 17 ND 10 SEQ ID NO:40 dots indicate identity; CpG dinucleotides are underlined .sub.1measured by ELISA using Quantikine kits from R&D Systems (pg/ml) Cells were cultured in 10% autologous serum with the indicated oligodeoxynucleotides (12 .mu.g/ml) for 4 hr in the case of TNF-.alpha. or 24 hr for the other cytokines before supernatant harvest and assay. Data are presented as the level of cytokine above that in wells with no added oligodeoxynucleotide.

CpG DNA Induces Cytokine Secretion by Human PBMC, Specifically Monocytes

The same panels of ODN used for studying mouse cytokine expression were used so determine whether human cells also are induced by CpG motifs to express cytokine (or proliferate), and to identify the CpG motif(s) responsible. Oligonucleotide 1619 (GTCGTT; SEQ. ID. NO: 57) was the best inducer of TNF-.alpha. and IFN-.gamma. secretion, and was closely followed by a nearly identical motif in oligonucleotide 1634 (GTCGCT; SEQ. ID. NO: 58) (Table 5). The motifs in oligodeoxynucleotides 1637 and 1614 (GCCGGT; SEQ. ID. NO: 109) and (GACGGT; SEQ. ID. NO: 110) led to strong IL-6 secretion with relatively little induction of other cytokines. Thus, it appears that human lymphocytes, like murine lymphocytes, secrete cytokines differentially in response to CpG dinucleotides, depending on the surrounding bases. Moreover, the motifs that stimulate murine cells best differ from those that are most effective with human cells. Certain CpG oligodeoxynucleotides are poor at activating human cells (oligodeoxynucleotides 1707, 1708, which contain the palindrome forming sequences GACGTC (SEQ. ID. NO: 111) and CACGTG (SEQ. ID. NO: 112) respectively).

The cells responding to the DNA appear to be monocytes, since the cytokine secretion is abolished by treatment of the cells with L-leucyl-L-leucine methyl ester (L-LME), which is selectively toxic to monocytes (but also to cytotoxic T lymphocytes and NK cells), and does not affect B cell Ig secretion (Table 6). The cells surviving L-LME treatment had >95% viability by trypan blue exclusion, indicating that the lack of a cytokine response among these cells did not simply reflect a nonspecific death of all cell types. Cytokine secretion in response to E. coli (EC) DNA requires unmethylated CpG motifs, since it is abolished by methylation of the EC DNA (next to the bottom row, Table 6). LPS contamination of the DNA cannot explain the results since the level of contamination was identical in the native and methylated DNA, and since addition of twice the highest amount of contaminating LPS had no effect (not shown).

TABLE-US-00010 TABLE 6 CpG DNA induces cytokine secretion by human PBMC TNF-.alpha. IL-6 IFN-.gamma. RANTES DNA (pg/ml).sup.1 (pg/ml) (pg/ml) (pg/ml) EC DNA (50 .mu.g/ml) 900 12,000 700 1560 EC DNA (5 .mu.g/ml) 850 11,000 400 750 EC DNA (0.5 .mu.g/ml) 500 ND 200 0 EC DNA (0.05 .mu.g/ml) 62.5 10,000 15.6 0 EC DNA (50 .mu.g/ml) + 0 ND ND ND L-LME.sub.2 EC DNA (10 .mu.g/ml) Methyl..sub.3 0 5 ND ND CT DNA (50 .mu.g/ml) 0 600 0 0 .sup.1Levels of all cytokines were determined by ELISA using Quantikine kits from R&D Systems as described in the previous table. Results are representative using PBMC from different donors. .sub.2Cells were pretreated for 15 min. with L-leucyl-L-leucine methyl ester (M-LME) to determine whether the cytokine production under these conditions was from monocytes (or other L-LME-sensitive cells). .sub.3EC DNA was methylated using 2 U/.mu.g DNA of CpG methylase (New England Biolabs) according to the manufacturer's directions, and methylation confirmed by digestion with Hpa-II and Msp-I. As a negative control, samples were included containing twice the maximal amount of LPS contained in the highest concentration of EC DNA which failed to induce detectable cytokine production under these experimental conditions. ND = not done

The loss of cytokine production in the PBMC treated with L-LME suggested that monocytes may be responsible for cytokine production in response to CpG DNA. To test this hypothesis more directly, the effects of CpG DNA on highly purified human monocytes and macrophages was tested. As hypothesized, CpG DNA directly activated production of the cytokines IL-6, GM-CSF, and TNF-.alpha. by human macrophages, whereas non-CpG DNA did not (Table 7).

TABLE-US-00011 TABLE 7 CpG DNA induces cytokine expression in purified human macrophages IL-6 (pg/ml) GM-CSF (pg/ml) TNF-.alpha. (pg/ml) Cells alone 0 0 0 CT DNA (50 .mu.g/ml) 0 0 0 EC DNA (50 .mu.g/ml) 2000 15.6 1000

Biological Role of IL-6 in Inducing Murine IgM Production in Response to CpG Motifs

The kinetic studies described above revealed that induction of IL-6 secretion, which occurs within 1 hr post CpG stimulation, precedes IgM secretion. Since the optimal CpG motif for ODN inducing secretion of IL-6 is the same as that for IgM (Table 2), whether the CpG motifs independently induce IgM and IL-6 production or whether the IgM production is dependent on prior IL-6 secretion was examined. The addition of neutralizing anti-IL-6 antibodies inhibited in vitro IgM production mediated by CpG ODN in a dose-dependent manner but a control antibody did not (FIG. 4A). In contrast, anti-IL-6, addition did not affect either the basal level or the CpG-induced B cell proliferation (FIG. 4B).

Increased Transcriptional Activity of the IL-6 Promoter in Response to CpG DNA

The increased level of IL-6 MnRNA and protein after CpG DNA stimulation could result from transcriptional or post-transcriptional regulation. To determine if the transcriptional activity of the IL-6 promoter was unregulated in B cells cultured with CpG ODN, a murine B cell line, WEHI-231, which produces IL-6 in response to CpG DNA, was transfected with an IL-6 promoter-CAT construct (pIL-6/CAT) (Pottrats, S. T. et al., 17B-estradiol) inhibits expression of human interleukin-6-promoter-reporter constructs by a receptor-dependent mechanism. J. Clin. Invest. 93:944). CAT assays were performed after stimulation with various concentrations of CpG or non-CpG ODN. As shown in FIG. 5, CpG ODN induced increased CAT activity in dose-dependent manner while non-CPG ODN failed to induce CAT activity. This confirms that CpG induces the transcriptional activity of the IL-6 promoter.

Dependence of B Cell Activation by CpG ODN on The Number of 5' and 3' Phosphorothioate Internucleotide Linkages

To determine whether partial sulfur modification of the ODN backbone would be sufficient to enhance B cell activation, the effects of a series of ODN with the same sequence, but with differing numbers of S internucleotide linkages at the 5' end of ODN were required to provide optimal protection of the ODN from degradation by intracellular exo- and endo-nucleases. Only chimeric ODN containing two 5' phosphorothioate-modified linkages, and a variable number of 3' modified linkages were therefore examined.

The lymphocyte stimulating effects of these ODN were tested at three concentrations (3.3, 10, and 30 .mu.M) by measuring the total levels of RNA synthesis (by .sup.3H uridine incorporation) or DNA synthesis (by .sup.3H thymidine incorporation) in treated spleen cell cultures (Example 10). O-ODN (0/0 phosphorothioate modifications) bearing a CpG motif caused no spleen cell stimulation unless added to the cultures at concentrations of at least 10 .mu.M (Example 10). However, when this sequence was modified with two S linkages at the 5' end and at least three S linkages at the 3' end, significant stimulation was seen at a dose of 3.3 .mu.M. At this low dose, the level of stimulation showed a progressive increase as the number of 3' modified bases was increased, until this reached or exceeded six, at which point the stimulation index began to decline. In general, the optimal number of 3' S linkages for spleen cell stimulation was five. Of all three concentrations tested in these experiments, the S-ODN was less stimulatory than the optimal chimeric compounds.

Dependent of GpG-Mediated Lymyhocyte Activation on The Type of Backbone Modification

Phosphorothioate modified ODN (S-ODN) are far more nuclease resistant than phosphodiester modified ODN (O-ODN). Thus, the increased immune stimulation caused by S-ODN and S-O-ODN (i.e., chimeric phosphorothioate ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are phosphorothioate modified) compared to O-ODN may result from the nuclease resistance of the former. To determine the role of ODN nuclease resistance in immune stimulation by CpG ODN, the stimulatory effects of chimeric ODN in which the 5' and 3' ends were rendered nuclease resistant with either methylphosphonate (MP-), methylphosphorothioate (MPS-), phosphorothioate (S-), or phosphorodithioate (S.sub.2-) internucleotide linkages were tested (Example 10). These studies showed that despite their nuclease resistance, MP-O-ODN were actually less immune stimulatory than O-ODN. However, combining the MP and S modifications by replacing both nonbridging O molecules with 5' and 3' MPS internucleotide linkages restored immune stimulation to a slightly higher level than that triggered by O-ODN.

S-O-ODN were far more stimulatory than O-ODN, and were even more stimulatory than S-ODN, at least at concentrations above 3.3 .mu.M. At concentrations below 3 .mu.M, the. S-ODN with the 3M sequence was more potent than the corresponding S-O-ODN, while the S-ODN with the 3D sequence was less potent than the corresponding S-O-ODN (Example 10). In comparing the stimulatory CpG motifs of these two sequences, it was noted that the 3D sequence is a perfect match for the stimulatory motif in that the CpG is flanked by two 5' purines and two 3' pyrimidines. However, the bases immediately flanking the CpG in ODN 3D are not optimal; it has a 5' pyrimidine and a 3' purine. Based on further testing, it was found that the sequence requirement for immune stimulation is more stringent for S-ODN than for S-O- or O-ODN. S-ODN with poor matches to the optimal CpG motif cause little or no lymphocyte activation (e.g., Sequence 3D). However, S-ODN with good matches to the motif, most critically at the positions immediately flanking the CpG, are more potent than the corresponding S-O-ODN (e.g., Sequence 3M, Sequences 4 and 6), even though at higher concentrations (greater than 3 .mu.M) the peak effect from the S-O-ODN is greater (Example 10).

S.sub.2-O-ODN were remarkably stimulatory, and caused substantially greater lymphocyte activation than the corresponding S-ODN or S-O-ODN at every tested concentration.

The increased B cell stimulation seen with CpG ODN bearing S or S.sub.2 substitutions could result from any or all of the following effects: nuclease resistance, increased cellular uptake, increased protein binding, and altered intracellular localization. However, nuclease resistance cannot be the only explanation, since the MP-O-ODN were actually less stimulatory than the O-ODN with CpG motifs. Prior studies have shown that ODN uptake by lymphocytes is markedly affected by the backbone chemistry (Zhao, et al. (1993) Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides. (Antisense Research and Development 3, 53-66; Zhao et al., (1994) Stage specific oligonucleotide uptake in murine bone marrow B cell precursors. Blood 84, 3660-3666). The highest cell membrane binding and uptake was seen with S-ODN, followed by S-O-ODN, O-ODN and MP-ODN. This differential uptake correlates with the degree of immune stimulation.

Unmethylated CpG Containing Oligos Have NK Cell Stimulatory Activity

Experiments were conducted to determine whether CpG containing oligonucleotides stimulated the activity of natural killer (NK) cells in addition to B cells. As shown in Table 8, a marked induction of NK activity among spleen cells cultured with CpG ODN 1 and 3Dd was observed. In contrast, there was relatively on induction in effectors that had been treated with non-CpG control ODN.

TABLE-US-00012 TABLE 8 Induction Of NK Activity By CpG Oligodeoxynucleotides (ODN) % YAC-1 Specific Lysis* % 2C11 Specific Lysis Effector: Target Effector: Target ODN 50:1 100:1 50:1 100:1 None -1.1 -1.4 15.3 16.6 1 16.1 24.5 38.7 47.2 3Dd 17.1 27.0 37.0 40.0 non-CpG ODN -1.6 -1.7 14.8 15.4

Induction of NK Activity by DNA Containing CpG Motifs, but Not by Non-CpG DNA.

Bacterial DNA cultured for 18 hrs at 37.degree. C. and then assayed for killing of K562 (human) or Yac-1 (mouse) target cells induced NK lytic activity in both mouse spleen cells depleted of B cells and human PBMC, but vertebrate DNA may be a consequence of its increased level of unmethylated CpG dinucleotides, the activating properties of more than 50 synthetic ODN containing unmethylated, methylated, or no CpG dinucleotides was tested. The results, summarized in Table 9, demonstrate that synthetic ODN can stimulate significant NK activity, as long as they contain at least one unmethylated CpG dinucleotide. No difference was observed in the stimulatory effects of ODN in which the CpG was within a palindrome (such as ODN 1585, which contains the palindrome AACGTT; SEQ. ID. NO: 105) from those ODN without palindromes (such as 1613 ro 1619), with the caveat that optimal stimulation was generally seem with ODN in which the CpG was flanked by two 5' purines or a 5' GpT dinucleotide and two 3 ' pyrimidines. Kinetic experiments demonstrated that NK activity peaked around 18 hrs. after addition of the ODN. The data indicates that the murine NK response is dependent on the prior activation of monocytes by CpG DNA, leading to the production of IL-12, TNF-.alpha., and IFN-.alpha./b (Example 11).

TABLE-US-00013 TABLE 9 Induction of NK Activity by DNA Containing CpG Motifs but not by Non-CpG DNA LU/10.sup.6 DNA or Cytokine Added Mouse Cells Human Cells Expt. 1 None 0.00 0.00 IL-2 16.68 15.82 E. Coli. DNA 7.23 5.05 Calf thymus DNA 0.00 0.00 Expt. 2 None 0.00 3.28 1585 ggGGTCAACGTTGAGggggg (SEQ ID No.12) 7.38 17.98 1629 --------------gtc------------- (SEQ ID No.41) 0.00 4.4 Expt. 3 None 0.00 1613 GCTAGACGTTAGTGT (SEQ ID No.42) 5.22 1769 --------------Z----------- (SEQ ID No.117) 0.02 ND 1619 TCCATGTCGTTCCTGATGCT (SEQ ID No.38) 3.35 1765 --------------Z------------------- (SEQ ID No.44) 0.11 CpG dinucleotides in ODN sequences are indicated by underlying; Z indicates methylcytosine. Lower case letters indicate nuclease resistant phosphorothioate modified internucleotide linkages which, in titration experiments, were more than 20 times as potent as non-modified ODN, depending on the flanking bases. Poly G ends (g) were used in some ODN, because they significantly increase the level of ODN uptake.

From all of these studies, a more complete understanding of the immune effects of CpG DNA has been developed, which is summarized in FIG. 6.

Immune activation by CpG motifs may depend on bases flanking the CpG, and the number of spacing of the CpGs present within an ODN. Although a single CpG in an ideal base context can be a very strong and useful immune activator, superior effects can be seen with ODN containing several CpGs with the appropriate spacing and flanking bases. For activation of murine B cells, the optimal CpG motif is TGACGTT (SEQ. ID. NO: 108); residues 10-17 of Seq. ID. No 70.

The following studies where conducted to identify optimal ODN sequences for to stimulation of human cells by examining the effects of changing the number, spacing, and flanking bases of CpG dinucleotides.

Identification of Phosohorothioate ODN with Optimal CpG Motifs for Activation of Human NK Cells.

To have clinical utility, ODN must be administered to a subject in a form that protects them against nuclease degradation. Methods to accomplish this with phosphodiester ODN are well known in the art and include encapsulation in lipids or delivery systems such as nanoparticles. This protection can also be achieved using chemical substitutions to the DNA such as modified DNA backbones including those in which the intemucleotide linkages are nuclease resistant. Some modifications may confer additional desirable properties such as increasing cellular uptake. For example, the phosphodiester linkage can be modified via replacement of one of the nonbridging oxygen atoms with a sulfur, which constitutes phosphorothioate DNA. Phosphorothioate ODN have enhanced cellular uptake (Krieg et al., Antisense Res. Dev. 6:133, 1996.) and improved B cell stimulation if they also have a CpG motif. Since NK activation correlates strongly with in vivo adjuvant effects, the identification of phosphorothioate ODN that will activate human NK cells is very important.

The effects of different phosphorothioate ODNs--containing CpG dinucleotides in various base contexts--on human NK activation (Table 10) were examined. ODN 1840, which contained 2 copies of the TGTCGTT (SEQ. ID. NO: 102) residues 14-20 of SEQ. ID. NO: 47 motif, had significant NK lytic activity (Table 10). To further identify additional ODNs optimal for NK activation, approximately one hundred ODN containing different numbers and spacing of CpG motifs, were tested with ODN1982 serving as a control. The results shown in Table 11.

Effective ODNs began with a TC or TG at the 5' end, however, this requirement was not mandatory. ODNs with internal CpG motifs (e.g., ODN 1840) are generally less potent stimulators than those in which a GTCGCT (SEQ. ID. NO: 58) motif immediately follows the 5' TC (e.g., ODN 1967 and 1968). ODN 1968, which has a second GTCGTT (SEQ. ID. NO: 57) motif in its 3' half, was consistently more stimulatory than ODN 1967, which lacks this second motif. ODN 1967, however, was slightly more potent than ODN 1968 in experiments 1 and 3, but not in experiment 2. ODN 2005, which has a third GTCGTT (SEQ. ID. NO: 57) motif, inducing slightly higher NK activity on average than 1968. However, ODN 2006, in which the spacing between the GTCGTT (SEQ. ID. NO: 57) motifs was increased by the addition of two Ts between each motif, was superior to ODN 2005 and to ODN 2007, in which only one of the motifs had the additional of the spacing two Ts. The minimal acceptable spacing between CpG motifs is one nucleotide as long as the ODN has two pyrimides (preferably T) at the 3' end (e.g., ODN 2015). Surprisingly, joining two GTCGTT (SEQ. ID. NO: 57) motifs end to end with a 5' T also created a reasonably strong inducer of NK activity (e.g., ODN 2016). The choice of thymine (T) separating consecutive CpG dinucleotides is not absolute, since ODN 2002 induced appreciable NK activation despite fact that adenine (A) separated its CpGs (i.e., CGACGTT; SEQ. ID. NO: 113). It should also be noted that ODNs containing no CpG (e.g., ODN 1982), runs of CpGs, or CpGs in bad sequence contents (e. e;. ODN 2010) had no stimulatory effect on NK activation.

TABLE-US-00014 TABLE 10 ODN Sequence (5'-3') LU cells 0.01 alone 1754 ACCATGGACGATCTGTTTCCCCTC 0.02 SEQ ID NO: 59 1758 TCTCCCAGCGTGCGCCAT 0.05 SEQ ID NO: 45 1761 TACCGCGTGCGACCCTCT 0.05 SEQ ID NO: 60 1776 ACCATGGACGAACTGTTTCCCCTC 0.03 SEQ ID NO: 61 1777 ACCATGGACGAGCTGTTTCCCCTC 0.05 SEQ ID NO: 62 1778 ACCATGGACGACCTGTTTCCCCTC 0.01 SEQ ID NO: 63 1779 ACCATGGACGTACTGTTTCCCCTC 0.02 SEQ ID NO: 64 1780 ACCATGGACGGTCTGTTTCCCCTC 0.29 SEQ ID NO: 65 1781 ACCATGGACGTTCTGTTTCCCCTC 0.38 SEQ ID NO: 66 1823 GCATGACGTTGAGCT 0.08 SEQ ID NO: 6 1824 CACGTTGAGGGGCAT 0.01 SEQ ID NO: 67 1825 CTGCTGAGACTGGAG 0.01 SEQ ID NO: 68 1828 TCAGCGTGCGCC 0.01 SEQ ID NO: 69 1829 ATGACGTTCCTGACGTT 0.42 SEQ ID NO: 70 1830 RANDOM SEQUENCE 0.25 1834 TCTCCCAGCGGGCGCAT 0.00 SEQ ID NO: 71 1836 TCTCCCAGCGCGCGCCAT 0.46 SEQ ID NO: 72 1840 TCCATGTCGTTCCTGTCGTT 2.70 SEQ ID NO: 73 1841 TCCATAGCGTTCCTAGCGTT 1.45 SEQ ID NO: 74

.sup.1Lytic units (LU) were measured as described (8). Briefly, PBMC were collected from normal donors and spun over Ficoll, then cultured with or without the indicated ODN (which were added to cultures at 6 .mu.g/ml) for 24 hr. Then their ability to lyse .sup.51Cr-labeled K562 cells was determined. The results shown are typical of those obtained with several different normal human donors. .sup.2This oligo mixture contained a random selection of all 4 bases at each position.

TABLE-US-00015 TABLE 11 Induction of NK LU by Phosphorothioate CpG ODN with Good Motifs ODN.sup.1 sequence (5'-3') SEQ ID NO: expt. 1 expt. 2 expt. 3 cells 0.00 1.26 0.46 alone 1840 TCCATGTCGTTCCTGTCGTT 73 2.33 ND ND 1960 TCCTGTCGTTCCTGTCGTT 77 ND 0.48 8.99 1961 TCCATGTCGTTTTTGTCGTT 78 4.03 1.23 5.08 1962 TCCTGTCGTTCCTTGTCGTT 52 ND 1.60 5.74 1963 TCCTTGTCGTTCCTGTCGTT 121 3.42 ND ND 1965 TCCTGTCGTTTTTTGTCGTT 53 0.46 0.42 3.48 1966 TCGTCGCTGTCTCCGCTTCTT 75 2.62 ND ND 1967 TCGTCGCTGTCTGCCCTTCTT 54 5.82 1.64 8.32 1968 TCGTCGCTGTTGTCGTTTCTT 55 3.77 5.26 6.12 1979.sup.2 TCCATGTZGTTCCTGTZGTT 122 1.32 ND ND 1982 TCCAGGACTTCTCTCAGGTT 79 0.05 ND 0.98 1990 TCCATGCGTGCGTGCGTTTT 80 2.10 ND ND 1991 TCCATGCGTTGCGTTGCGTT 81 0.89 ND ND 2002 TCCACGACGTTTTCGACGTT 82 4.02 1.31 9.79 2005 TCGTCGTTGTCGTTGTCGTT 47 ND 4.22 12.75 2006 TCGTCGTTTTGTCGTTTTGTCGT 123 ND 6.17 12.82 2007 TCGTCGTTGTCGTTTTGTCGTT 49 ND 2.68 9.66 2008 GCGTGCGTTGTCGTTGTCGTT 56 ND 1.37 8.15 2010 GCGGCGGGCGGCGCGCGCCC 83 ND 0.01 0.05 2012 TGTCGTTTGTCGTTTGTCGTT 48 ND 2.02 11.61 2013 TGTCGTTGTCGTTGTCGTTGTCGTT 84 ND 0.56 5.22 2014 TGTCGTTGTCGTTGTCGTT 50 ND 5.74 10.89 2015 TCGTCGTCGTCGTT 51 ND 4.53 10.13 2016 TGTCGTTGTCGTT 85 ND 6.54 8.06 .sup.1PBMC essentially as described herein. Results are representative of 6 separate experiments; each experiment represents a different donor. .sup.2This is the methylated version of ODN 1840; Z = 5-methyl cytosine LU is lytic units; ND = not done; CpG dinucleotides are underlined for clarity.

Identification of Phosphorothioate ODN with Optimal CpG Motifs for Activation of Human B Cell Proliferation.

The ability of a CpG ODN to induce B cell proliferation is a good measure of its adjuvant potential. Indeed, ODN with strong adjuvant effects generally also induce B cell proliferation. To determine whether the optimal CpG ODN for inducing B cell proliferation are the same as those for inducing NK cell activity, similar panels of ODN (Table 12) were tested. The most consistent stimulation appeared with ODN 2006 (Table 12).

TABLE-US-00016 TABLE 12 Induction of human B cell proliferation by Phosphorothioate CpG ODN Stimulation Index.sup.1 ODN sequence (5'-3') SEQ ID NO: expt. 1 expt. 2 expt. 3 expt. 4 expt. 5 expt. 6 1840 TCCATGTCGTTCCTGTCGTT 73 4 ND ND ND ND 34 1841 TCCATAGCGTTCCTAGCGTT 74 3 ND ND ND ND ND 1960 TCCTGTCGTTCCTGTCGTT 77 ND 2.0 2.0 3.6 ND ND 1961 TCCATGTCGTTTTTGTCGTT 78 2 3.9 1.9 3.7 ND 37 1962 TCCTGTCGTTCCTTGTCGTT 52 ND 3.8 1.9 3.9 5.4 35 1963 TCCTTGTCGTTCCTGTCGTT 121 3 ND ND ND ND ND 1965 TCCTGTCGTTTTTTGTCGTT 53 4 3.7 2.4 4.7 6.0 43 1967 TCGTCGCTGTCTGCCCTTCTT 54 ND 4.4 2.0 4.5 5.0 36 1968 TCGTCGCTGTTGTCGTTTCTT 55 ND 4.0 2.0 4.9 8.7 38 1982 TCCAGGACTTCTCTCAGGTT 79 3 1.8 1.3 3.1 3.2 12 2002 TCCACGACGTTTTCGACGTT 86 ND 2.7 1.4 4.4 ND 14 2005 TCGTCGTTGTCGTTGTCGTT 47 5 3.2 1.2 3.0 7.9 37 2006 TCGTCGTTTTGTCGTTTTGTCGTT 46 4 4.5 2.2 5.8 8.3 40 2007 TCGTCGTTGTCGTTTTGTCGTT 49 3 4.0 4.2 4.1 ND 22 2008 GCGTGCGTTGTCGTTGTCGTT 56 ND 3.0 2.4 1.6 ND 12 2010 GCGGCGGGCGGCGCGCGCCC 83 ND 1.6 1.9 3.2 ND ND 2012 TGTCGTTTGTCGTTTGTCGTT 48 2 2.8 0 3.2 ND 33 2013 TGTCGTTGTCGTTGTCGTTGTCGTT 84 3 2.3 3.1 2.8 ND 7 2014 TGTCGTTGTCGTTGTCGTT 50 3 2.5 4.0 3.2 6.7 14 2015 TCGTCGTCGTCGTT 51 5 1.8 2.6 4.5 9.4 1 2016 TGTCGTTGTCGTT 85 ND 1.1 1.7 2.7 7.3 1 .sup.1Cells = human spleen cells stored at -70.degree. C. after surgical harvest or PBMC collected from normal donors and spun over Ficoll. Cells were cultured in 96 well U-bottom microtiter plates with or without the indicated ODN (which were added to cultures at 6 .mu.ml). N = 12 experiments. Cells were cultured for 4-7 days, pulsed with 1 .mu.Ci of .sup.3H thymidine for 18 hr. before harvest and scintillation counting. Stimulation index = the ratio of cpm in wells without ODN to that in wells that had been stimulated throughout the culture period with the indicated ODN (there were no further additions of ODN after the cultures were set up). ND = not done.

Identification of Phosphorothioate ODN that Induce Human IL-12 Secretion

The ability of a CpG ODN to induce IL-12 secretion is a good measure of its adjuvant potential, especially in terms of its ability to induce a Th1 immune response, which is highly dependent on IL-12. Therefore, the ability of a panel of phosphorothioate ODN to induce 0IL-12 secretion from human PBMC in vitro (Table 13) was examined. These experiments showed that in some human PBMC, most CpG ODN could induce IL-12 secretion (e.g., expt. 1). However, other donors responded to just a few CpG ODN (E.g., expt. 2.). ODN 2006 was a consistent inducer of IL12 secretion from most subjects (Table 13).

TABLE-US-00017 TABLE 13 Induction of human IL-12 secretion by Phosphorothioate CpG ODN IL-12 (pg/ml) ODN.sup.1 sequence (5'-3') SEQ ID NO: expt. 1 expt. 2 cells alone 0 0 1962 TCCTGTCGTTCCTTGTCGTT 52 19 0 1965 TCCTGTCGTTTTTTGTCGTT 53 36 0 1967 TCGTCGCTGTCTGCCCTTCTT 119 41 0 1968 TCGTCGCTGTTGTCGTTTCTT 120 24 0 2005 TCGTCGTTGTCGTTGTCGTT 47 25 0 2006 TCGTCGTTTTGTCGTTTTGTCGTT 46 29 15 2014 TGTCGTTGTCGTTGTCGTT 50 28 0 2015 TCGTCGTCGTCGTT 51 14 0 2016 TGTCGTTGTCGTT 85 3 0 .sup.1PBMC were collected from normal donors and spun over Ficoll, then cultured at 10.sup.6 cells/well in 96 well microtiter plates with or without the indicated ODN which were added to cultures as .mu.g/ml. Supernatants were collected at 24 hr and tested for IL-12 levels by ELISA as described methods. A standard curve was run in each experiment, which represents a different donor.

Identification of B cell and Monocyte/NK Cell-specific Oligonucleotides

As shown in FIG. 6, CpG DNA can directly activate highly purified B cells and monocytic cells. There are many similarities in the mechanism through which CpG DNA activates these cell types. For example, both require NFkB activation as explained further below.

In further studies of different immune effects of CpG DNA, it was found that there is more than one type of CpG motif. Specifically, olio 1668, with the best mouse B cell motif, is a strong inducer of both B cell and natural killer (NK) cell activation, while olio 1758 is a weak B cell activator, but still induces excellent NK responses (Table 14).

TABLE-US-00018 TABLE 14 Different CpG motifs stimulate optimal murine B cell and NK activation B cell NK ODN Sequence activation.sup.1 activation.sup.2 1668 TCCATGACGTTCCTGATGCT (SEQ ID. NO. 7) 42,849 2.52 1758 TCTCCCAGCGTGCGCCAT (SEQ ID. NO. 45) 1,747 6.66 NONE 367 0.00 CpG dinucleotides are underlined; oligonucleotides were synthesized with phosphorothioate modified backbones to improve their nuclease resistance. .sup.1Measured by .sup.3H thymidine incorporation after 48 hr culture with oligodeoxynucleotides at a 200 nM concentration as described in Example 1. .sup.2Measured in lytic units.

Teleological Basis of Immunostimulatory Nucleic Acids

Vertebrate DNA is highly methylated and CpG dinucleotides are under represented. However, the stimulatory CpG motif is common in microbial genomic DNA, but quite rare in vertebrate DNA. In addition, bacterial DNA has been reported to induce B cell proliferation and immunoglobulin (Ig) production, while mammalian DNA does not (Messina, J. P. et al., J. Immunol. 147:1759 (1991)). Experiments further described in Example 3, in which methylation of bacterial DNA with CpG methylase was found to abolish mitogenicity, demonstrates that the difference in CpG status is the cause of B cell stimulation by bacterial DNA. This data supports the following conclusion: that unmethylated CpG dinucleotides present within bacterial DNA are responsible for the stimulatory effects of bacterial DNA.

Teleologically, it appears likely that lymphocyte activation by the CpG motif represents an immune defense mechanism that can thereby distinguish bacterial from host DNA. Host DNA, which would commonly be present in many anatomic regions and areas of inflammation due to apoptosis (cell death), would generally induce little or no lymphocyte activation due to CpG suppression and methylation. However, the presence of bacterial DNA containing unmethylated CpG motifs can cause lymphocyte activation precisely in infected anatomic regions, where it is beneficial. This novel activation pathway provides a rapid alternative to T cell dependent antigen specific B cell activation. Since the CpG pathway synergizes with B cell activation through the antigen receptor, B cells bearing antigen receptor specific for bacterial antigens would receive on e activation signal through cell membrane Ig and a second signal from bacterial DNA, and would therefore tend to be preferentially activated. The interrelationship of this pathway with other pathways of B cell activation provide a physiologic mechanism employing a polyclonal antigen to induce antigen-specific responses.

However, it is likely that B cell activation would not be totally nonspecific. B cells bearing antigen receptors specific for bacterial products could receive one activation signal through cell membrane Ig, and a second from bacterial DNA, thereby more vigorously triggering antigen specific immune responses. As with other immune defense mechanisms, the response to bacterial DNA could have undesirable consequences in some settings. For example, autoimmune responses to self antigens would also tend to be preferentially triggered by bacterial infections, since autoantigens could also provide a second activation signal to autoreactive B cells triggered by bacterial DNA. Indeed the induction of autoimmunity by bacterial infections is a common clinical observance. For example, the autoimmune disease systemic lupus erythematosus, which is: i) characterized by the production of anti-DNA antibodies; ii) induced by drugs which inhibit DNA methyltransferase (Cornaccia, E. J. et al., J. Clin. Invest. 92:38 (1993)); and iii) associated with reduced DNA methylation (Richardson, B., L. et al., Arth. Rheum 35:647 (1992)), is likely triggered at least in part by activation of DNA-specific B cells through stimulatory signals provided by CpG motifs, as well as by binding of bacterial DNA to antigen receptors.

Further, sepsis, which is characterized by high morbidity and mortality due to massive and nonspecific activation of the immune system may be initiated by bacterial DNA and other products released from dying bacteria that reach concentrations sufficient to directly activate many lymphocytes. Further evidence of the role of CpG DNA in the sepsis syndrome is described in Cowdery, J., et. al., (1996) the Journal of Immunology 156:4570-4575.

Unlike antigens that trigger B cells through their surface Ig receptor, CpG-ODN did not induce any detectable Ca.sup.2+ flux, changes in protein tyrosine phosphorylation, or IP 3 generation. Flow cytometry with FITC-conjugated ODN with or without a CpG motif was performed as described in Zhao, Q et al., (Antisense Research and Development 3:53-66 (1993)), and showed equivalent membrane binding, cellular uptake, efflux, and intracellular localization. This suggests that there may not be cell membrane proteins specific for CpG ODN. Rather than acting through the cell membrane, that data suggests that unmethylated CpG containing oligonucleotides require cell uptake for activity: ODN covalently linked to a solid Teflon support were nonstimulatory, as were biotinylated ODN immobilized on either avidin beads or avidin coated petri dishes. CpG ODN conjugated to either FITC or biotin retained full mitogenic properties, indicated no stearic hindrance.

Recent data indicate the involvement of the transcription factor NFkB as a direct or indirect mediator of the CpG effect. For example, within 15 minutes of treating B cells or monocytes with CpG DNA, the level of NFkB binding activity is increased (FIG. 7). However, it is not increased by DNA that does not contain CpG motifs. In addition, it was found that two different inhibitors of NFkB activation, PDTC and gliotoxin, completely block the lymphocyte stimulation by CpG DNA as measured by B cell proliferation or monocytic cell cytokine secretion, suggesting that NFkB activation is required for both cell types.

There are several possible mechanisms through which NFkB can be activated. These include through activation of various protein kinases, or through the generation of reactive oxygen species. No evidence for protein kinase activation induced immediately after CpG DNA treatment of B cells or monocytic cells have been found, and inhibitors of protein kinase A, protein kinase C, and protein tyrosine kinases had no effects on the CpG induced activation. k However, CpG DNA causes a rapid induction of the production of reactive oxygen species in both B cells and monocytic cells, as detected by the sensitive fluorescent dye dihydrorhodamine 123 as described in Royall, J. A., and Ischiropoulos, H. (Archives of Biochemistry and Biophysics 302:348-355 (1993)). Moreover, inhibitors of the generation of these reactive oxygen species completely block the induction of NFkB and the later induction of cell proliferation and cytokine secretion by CpG DNA.

Work backwards, the next question was how CpG DNA leads to the generation of reactive oxygen species so quickly. Previous studies by the inventors demonstrated that oligonucleotides and plasmid or bacterial DNA are taken up by cells into endosomes. These endosomes rapidly become acidified inside the cell. To determine whether this acidification step may be important in the mechanism through which CpG DNA activates reactive oxygen species, the acidification step was blocked with specific inhibitors of endosome acidification including chloroquine, monensin, and bafilomycin, which work through different mechanisms. FIG. 8A shows the results from a flow cytometry study using mouse B cells with the dihydrorhodamine 123 dye to determine levels of reactive oxygen species. The dye only sample in Panel A of the figure shows the background level of cells positive for the dye at 28.6%. as expected, this level of reactive oxygen species was greatly increased to 80% in the cells treated for 20 minutes with PMA and ionomycin, a positive control (Panel B). The cells treated with the CpG oligo also showed an increase in the level of reactive oxygen species such that more than 50% of the cells became positive (Panel D). However, cells treated with an oligonucleotide with the identical sequence except that the CpG was switched did not show this significant increase in the level of reactive oxygen species (Panel E).

In the presence of chloroquine, the results are very different (FIG. 8B). Chloroquine slightly lowers the background level of reactive oxygen species in the cells such that the untreated cells in Panel A have only 4.3% that are positive. Chloroquine completely abolishes the induction of reactive oxygen species in t he cells treated with CpG DNA (Panel B) but does not reduce the level of reactive oxygen species in t he cells treated with PMA and ionomycin (Panel E). This demonstrates that unlike the PMA plus ionomycin, the generation of reactive oxygen species following treatment of B cells with CpG DNA requires that the DNA undergo an acidification step in the endosomes. This is a completely novel mechanism of leukocyte activation. Chloroquine, monensin, and bafilomycin also appear to block the activation of NFkB by CpG DNA as well as the subsequent proliferation and induction of cytokine secretion.

Chronic Immune Activation by CpG DNA and Autoimmune Disorders

B cell activation by CpG DNA synergizes with signals through the B cell receptor. This raises the possibility that DNA-specific B cells may be activated by the concurrent binding of bacterial DNA to their antigen receptor, and by the co-stimulatory CpG-mediated signals. In addition, CpG DNA induces B cells to become resistant to apoptosis, a mechanism thought to be important for preventing immune responses to self antigens, such as DNA. Indeed, exposure to bDNA can trigger anti-DNA Ab production. Given this potential ability of CpG DNA to promote autoimmunity, it is therefore noteworthy that patients with the autoimmune disease systemic lupus erythematosus have persistently elevated levels of circulating plasma DNA which is enriched in hypomethylated CpGs. These findings suggest a possible role for chronic immune activation by CpG DNA in lupus etiopathogenesis.

A class of medications effective in the treatment of lupus is antimalarial drugs, such as chloroquine. While the therapeutic mechanism of these drugs has been unclear, they are known to inhibit endosomal acidification. Leukocyte activation by CpG DNA is not medicated through binding to a cell surface receptor, but requires cell uptake, which occurs via adsorptive endocytosis into an acidified chloroquine-sensitive intracellular compartment. This suggested the hypothesis that leukocyte activation by CpG DNA may occur in association with acidified endosomes, and might even be pH dependent. To test this hypothesis specific inhibitors of DNA acidification were applied to determine whether B cells or monocytes could respond to CpG DNA if endosomal acidification was prevented.

The earliest leukocyte activation event that was detected in response to CpG DNA is the production of reactive oxygen species (ROS), which is induced within five minutes in primary spleen cells and both B and monocyte cell lines. Inhibitors of endosomal acidification including chloroquine, bafilomycin A, and monensin, which have different mechanisms of action, blocked the CpG-induced generation of ROS, but had no effect on ROS generation mediated by PMA, or ligation of CD40 or IgM. These studies show that ROS generation is a common event in leukocyte activation through diverse pathways. This ROS generation is generally independent of endosomal acidification, which is required only for the ROS response to CpG DNA. ROS generation in response to CpG is not inhibited by the NF.kappa.B inhibitor gliotoxin, confirming that it is not secondary to NF.kappa.B activation.

To determine whether endosomal acidification of CpG DNA was also required for its other immune stimulatory effects were performed. Both LPS and CpG DNA induce similar rapid NF.kappa.B activation, increases in proto-oncogene mRNA levels, and cytokine secretion. Activation of NF.kappa.B by DNA depended on CpG motifs since it was not induced by bDNA treated with CpG methylase, nor by ODN in which bases were switched to disrupt the CpGs. Supershift experiments using specific antibodies indicated that the activated NF.kappa.B complexes included the p50 and p65 components. Not unexpectedly, NF.kappa.B activation in LPS- or CpG-treated cells was accompanied by the degradation of I.kappa.B.alpha. and I.kappa.B.beta.. However, inhibitors of endosomal acidification selectively blocked all of the CpG-induced but none of the LPS-induced cellular activation events. The very low concentration of chloroquine (<10 .mu.M) that has been determined to inhibit CpG-mediated leukocyte activation is noteworthy since it is well below that required for antimalarial activity and other reported immune effects (e.g., 100-1000 .mu.M). These experiments support the role of a pH-dependent signaling mechanism in mediating the stimulatory effects of CpG DNA.

TABLE-US-00019 TABLE 15 Specific blockade of CpG-induced TNF-.alpha. and IL-12 expression by inhibitors of endosomal acidification or NF.kappa.B activation Inhibitors: Bafilomycin Chloroquine Monensin NAC TPCK Gliotoxin Bisglioxin Medium (250 nM) (2.5 .mu.g/ml) (10 .mu.M) (50 mM) (50 .mu.M) (0.1 .mu.g/ml) (0.1 .mu.g/ml) activators TNF-.alpha. IL-12 TNF-.alpha. IL-12 TNF-.alpha. IL-12 TNF-.alph- a. IL-12 TNF-.alpha. TNF-.alpha. TNF-.alpha. TNF-.alpha. Medium 37 147 46 102 27 20 22 73 10 24 17 41 CpG 455 17,114 71 116 28 6 49 777 54 23 31 441 ODN LPS 901 22,485 1370 4051 1025 12418 491 4796 417 46 178 1120 Table 15 legend IL-12 and TNF-.alpha. assays: The murine monocyte cell line J774 (1 .times. 10.sup.5 cells/ml for IL-12 or 1 .times. 10.sup.6 cells/ml for TNF-.alpha.), were cultured with or without the indicated inhibitors at the concentrations shown for 2 hr and then stimulated with the CpG oligodeoxynucleotide (ODN) 1826 (TCCATGACGTTCCTGACGTT SEQ ID NO: 10) at 2 .mu.M or LPS (10 .mu.g/ml) for 4 hr (TNF-.alpha.) or 24 hr (IL-12) at which time the supernatant was harvested. ELISA for IL-12 or TNF-.alpha. (pg/ml) was performed on the supernatants essentially as described (A. K. Krieg, A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. Koretzky and D. Klinman, Nature 374, 546 (1995); Yi, A.-K., D. M. Klinman, T. L. Martin, S. Matson and A. M. Krieg, J. Immunol., 157, 5394-5402 (1996); Krieg, A. M, J. Lab. Clin. Med., 128, 128-133 (1996). Cells cultured with ODN that lacked CpG motifs did not induce cytokine secretion. Similar specific inhibition of CpG responses was seen with IL-6 assays, and in experiments using primary spleen cells or the B cell lines CH12.LX and WEHI-231. 2.5 .mu.g/ml of chloroquine is equivalent to <5 .mu.M. Other inhibitors of NF-.kappa.B activation including PDTC and calpain inhibitors I and II gave similar results to the inhibitors shown. The results shown are representative of those obtained in ten different experiments.

Excessive immune activation by CpG motifs may contribute to the pathogenesis of the autoimmune disease systemic lupus erythematosus, which is associated with elevated levels of circulating hypomethylated CpG DNA. Chloroquine and related antimalarial compounds are effective therapeutic agents for the treatment of systemic lupus erythematosus and some other autoimmune diseases, although their mechanism of action has been obscure. Our demonstration of the ability of extremely low concentrations of chloroquine to specifically inhibit CpG-mediated leukocyte activation suggests a possible new mechanism for its beneficial effect. It is noteworthy that lupus recurrences frequently are thought to be triggered by microbial infection. Levels of bDNA present in infected tissues can be sufficient to induce a local inflammatory response. Together with the likely role of CpG DNA as a mediator of the sepsis syndrome and other diseases our studies suggest possible new therapeutic applications for the antimalarial drugs that act as inhibitors of endosomal acidification.

CpG-induced ROS generation could be an incidental consequence of cell activation, or a signal that mediates this activation. The ROS scavenger N-acetyl-L-cysteine (NAC) blocks CpG-induced NF.kappa.B activation, cytokine production, and B cell proliferation, suggesting a casual role for ROS generation in these pathways. These data are compatible with previous evidence supporting a role for ROS in the activation of NF.kappa.B. WEHI-231 B cells (5.times.10.sup.5 cells/ml) were precultured for 30 minutes with or without chloroquine (5 .mu.g/ml [<10 .mu.M]) or gliotoxin (0.2 .mu.g/ml). Cell aliquots were then cultured as above for 10 minutes in RPMI medium with or without a CpG ODN (1826) or non-CpG ODN (1911) at 1 .mu.M or phorbol myristate acetate (PMA) plus ionomycin (iono). Cells were then stained with dihydrorhodamine-123 and analyzed for intracellular ROS production by flow cytometry as described (A. K. Krieg, A.-K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. Koretzky and D. Klinman, Nature 374, 546 (1995); Yi, A.-K., D. M. Klinman, T. L. Martin, S. Matson and A. M. Krieg, J. Immunol., 157, 5394-5402 (1996); Krieg, A. M, J. Lab. Clin. Med., 128, 128-133 (1996)). J1774 cells, a monocytic line, showed similar pH-dependent CpG induced ROS responses. In contrast, CpG DNA did not induce the generation of extracellular ROS, nor any detectable neutrophil ROS. The concentrations of chloroquine (and those used with the other inhibitors of endosomal acidification) prevented acidification of the internalized CpG DNA using fluorescein conjugated ODN as described by Tonkinson, et al., (Nucl. Acids Res. 22, 4268 (1994); A. M. Krieg, In: Delivery Strategies for Antisense Oligonucleotide Therapeutics. Editor, S. Akhtar, CRC Press, Inc., pp. 177(1995)). At higher concentrations than those required to inhibit endosomal acidification, nonspecific inhibitory effects were observed. Each experiment was performed at least three times with similar results.

While NF.kappa.B is known to be an important regulator of gene expression, it's role in the transcriptional response to CpG DNA was uncertain. To determine whether this NF.kappa.B activation was required for the CpG mediated induction of gene expression cells were activated with CpG DNA in the presence or absence of pyrrolidine dithiocarbamate (PDTC), an inhibitor of I.kappa.B phosphorylation. These inhibitors of NF.kappa.B activation completely blocked the CpG-induced expression of protooncogene and cytokine mRNA and protein, demonstrating the essential role of NF.kappa.B as a mediator of these events. None of the inhibitors reduced cell viability under the experimental conditions used in these studies. A J774, a murine monocyte cell line, was cultured in the presence of calf thymus (CT), E. Coli (EC), or methylated E. Coli (mEC) DNA (methylated with CpG methylase as .sup.described) at 5 .mu.g/ml or a CpG oligodeoxynucleotide (ODN 1826; Table 15) or a non-CpG ODN (ODN 1745; TCCATGAGCTTCCTGAGTCT, SEQ. ID. NO: 8) at 0.75 .mu.M for 1 hr, following which the cells were lysed and nuclear extracts prepared. A double stranded ODN containing a consensus NF.kappa.B site was 5' radiolabeled and used as a probe for EMSA essentially as described (J. D. Dignam, R. M. Lebovitz and R. G. Roeder, Nucleic Acids Res. 11, 1475 (1983); M. Briskin, M. Damore, R. Law, G. Lee, P. W. Kincade, C. H. Sibley, M. Kuehl and R. Wall, Mol. Cell. Biol. 10, 422 (1990)). The position of the p50/p65 heterodimer was determined by supershifting with specific Ab to p65 and p50 (Santa Cruz Biotechnology, Santa Cruz, Calif.). Chloroquine inhibition of CpG-induced but not LPS-induced NF.kappa.B activation was established using J774 cells. The cells were precultured for 2 hr in the presence or absence of chloroquine (20 .mu.g/ml) and then stimulated as above for 1 hr with either EC DNA, CpG ODN, non-CpG ODN or LPS (1 .mu.g/ml). Similar chloroquine sensitive CpG-induced activation of NF.kappa.B was seen in a B cell line, WEHI-231 and primary spleen cells. These experiments were performed three times over a range of chloroquine concentrations form 2.5 to 20 .mu.g/ml with similar results.

It was also established that CpG-stimulated mRNA expression requires endosomal acidification and NF.kappa.B activation in B cells and monocytes. J774 cells (2.times.10.sup.6 cells/ml) were cultured for 2 hr in the presence or absence of chloroquine (2.5 .mu.g/ml [<5 .mu.M]) or N-tosyl-L-phenylalanine chlorometryl ketone (TPCK: 50 .mu.M), a serine/threonine protease inhibitor that prevents I.kappa.B proteolysis and thus blocks NF.kappa.B activation. Cells were then stimulated with the addition of E. Coli DNA (EC: 50 .mu.g/ml), calf thymus DNA (CT: 50 .mu.g/ml), LPS (10 .mu.g/ml), CpG ODN (1826; 1 .mu.M), or control non CpG ODN (1911; 1 .mu.M) for 3 hr. WEHI-231 B cells (5.times.10.sup.5 cells/ml) were cultured in the presence or absence of gliotoxin (0.1 .mu.g/ml) or bisgliotoxin (0.1 .mu.g/ml) for 2 hrs and then stimulated with a CpG ODN (1826), or control non-CpG ODN (1911; TCCAGGACTTTCCTCAGGTT, SEQ. ID. NO. 97) at 0.5 .mu.M for 8 hr. In both cases, cells were harvested and RNA was prepared using RNAzol following the manufacturer's protocol. Multi-probe RNASE protection assay was performed as described (A.-K. Yi, P. Hornbeck, D. E. Lafrenz and A. M. Krieg, J. Immunol., 157, 4918-4925 (1996). Comparable amounts of RNA were loaded into each lane by using ribosomal MRNA as a loading control (L32). These experiments were performed three times with similar results.

The results indicate that leukocytes respond to CpG DNA through a novel pathway involving the pH-dependent generation of intracellular ROS. The pH dependent step may be the transport or processing of the CpG DNA, the ROS generation, or some other event. ROS are widely thought to be second messengers in signaling pathways in diverse cell types, but have not previously been shown to mediate a stimulatory signal in B cells.

Presumably, there is a protein in or near the endosomes that specifically recognizes DNA containing CpG motifs and leads to the generation of reactive oxygen species. To detect any protein in the cell cytoplasm that may specifically bind CpG DNA, electrophoretic mobility shift assays (EMSA) were used with 5' radioactively labeled oligonucleotides with or without CpG motifs. A band was found that appears to represent a protein binding specifically to single stranded oligonucleotides that have CpG motifs, but not to oligonucleotides that lack CpG motifs or to oligonucleotides in which the CpG motif has been methylated. This binding activity is blocked if excess of oligonucleotides that contain the NF.kappa.B binding site was added. This suggests that an NF.kappa.B or related protein is a component of a protein or protein complex that binds the stimulatory CpG oligonucleotides.

No activation of CREB/ATF proteins was found at time points where NF.kappa.B was strongly activated: These data therefore do not provide proof the NF.kappa.B proteins actually bind to the CpG nucleic acids, but rather that the proteins are required in some way for the CpG activity. It is possible that a CREB/ATF or related protein may interact in some way with NF.kappa.B proteins or other proteins thus explaining the remarkable similarity in the binding motifs for CREB proteins and the optimal CpG motif. It remains possible that the oligos bind to a CREB/ATF or related protein, and that this leads to NF.kappa.B activation.

Alternatively, it is very possible that the CpG nucleic acids may bind to one of the TRAF proteins that bind to the cytoplasmic region of CD40 and mediate NF.kappa.B activation when CD40 is cross-linked. Examples of such TRAF proteins include TRAF-2 and TRAF-5.

Method for Making Immunostimulatory Nucleic Acids

For use in the instant invention, nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (S. L. Beaucage and M. H. Caruthers, (1981) Tet. Let. 22:1859); nucleoside H-phosphonate method (Garegg et al., (1986) Tet. Let. 27:4051-4054; Froehler et al., (1986) Nucl. Acid. Res 14:5399-5407; Garegg eg al., (1986) Tet. Let. 27:4055-4058, Gaffney et al., (1988) Tet. Let. 29:2619-2622). These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. Alternatively, oligonucleotides can be prepared from existing nucleic acid sequences (e.g. genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.

For use in vivo, nucleic acids are preferably relatively resistant to degradation (e.g. via endo- and exo-nucleases). Secondary structures, such as stem loops, can stabilize nucleic acids against degradation. Alternatively, nucleic acid stabilization can be accomplished via phosphate backbone modifications. A preferred stabilized nucleic acid has at least a partial phosphorothioate modified backbone. Phosphorothioates may be synthesized using automated techniques employing either phosphoramidate or H-phosphonate chemistries. Aryl- and alkyl-phosphonates can be made e.g as described in U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which the charged oxygen moiety is alkylated as described in U.S. Pat. No. 5,023,243 and European Patent No. 092,574) can be prepared by automated solid phase synthesis using commercially available reagents. Methods for making other DNA backbone modifications and substitutions have been described (Uhlmann, E. And Peyman, A. (1990) Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165). 2'-O-methyl nucleic acids with CpG motifs also cause immune activation, as do ethoxy-modified CpG nucleic acids. In fact, no backbone modifications have been found that completely abolish the CpG effect, although it is greatly reduced by replacing the C with a 5-methyl C.

For administration in vivo, nucleic acids may be associated with a molecule that results in higher affinity binding to target cell (e.g. B-cell, monocytic cell and natural killer (NK) cell) surfaces and/or increased cellular uptake by target cells to form a "nucleic acid delivery complex". Nucleic acids can be ionically, or covalently associated with appropriate molecules using techniques which are well known in the art. A variety of coupling or crosslinking agents can be sued e.g. Protein A, carbodiimide, and N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP). Nucleic acids can alternatively be encapsulated in liposomes or virosomes using well-known techniques.

Therapeutic Uses of Immunostimulatory Nucleic Acid Molecules

Based on their immunostimulatory properties, nucleic acid molecules containing at least one unmethylated CpG dinucleotide can be administered to a subject in vivo to treat an "immune system deficiency". Alternatively, nucleic acid molecules containing at least one unmethylated CpG dinucleotide can be contacted with lymphocytes (e.g. B cells, monocytic cells or NK cells) obtained from a subject having an immune system deficiency ex vivo and activated lymphocytes can then be re-implanted in the subject.

As reported herein, in response to unmethylated CpG containing nucleic acid molecules, an increased number of spleen cells secrete IL-6, IL-12, IFN.gamma., IFN-.alpha., IFN-.beta., IL-1, IL-3, IL-10, TNF-.alpha., TNF-.beta., GM-CSF, RANTES, and probably others. The increased IL-6 expression was found to occur in B cells, CD4.sup.+T cells and monocytic cells.

Immunostimulatory nucleic acid molecules can also be administered to a subject in conjunction with a vaccine to boost a subject's immune system and thereby effect a better response from the vaccine. Preferably the immunostimulatory nucleic acid molecule is administered slightly before or at the same time as the vaccine. A conventional adjuvant may optionally be administered in conjunction with the vaccine, which is minimally comprised of an antigen, as the conventional adjuvant may further improve the vaccination by enhancing antigen absorption.

When the vaccine is a DNA vaccine at least two components determine its efficacy. First, the antigen encoded by the vaccine determines the specificity of the immune response. Second, if the backbone of the plasmid contains CpG motifs, its functions as an adjuvant for the vaccine. Thus, CpG DNA acts as an effective "danger signal" and causes the immune system to respond vigorously to new antigens in t he area This mode of action presumably results primarily from the stimulatory local effects of CpG DNA on dendritic cells and other "professional" antigen presenting cells, as well as from the co-stimulatory effects on B cells.

Immunostimulatory oligonucleotides and unmethylated CpG containing vaccines, which directly activate lymphocytes and co-stimulate an antigen-specific response, are fundamentally different from conventional adjuvants (e.g. aluminum precipates), which are inert when injected alone and are thought to work through absorbing the antigen and thereby presenting it more effectively to immune cells. Further conventional adjuvants only work for certain antigens, only induce an antibody (humoral) immune response (Th2), and are very poor at inducing cellular immune responses (Th1). For many pathogens, the humoral response contributes little to protection, and can even be detrimental.

In addition, an immunostimulatory oligonucleotide can be administered prior to along with or after administration of a chemotherapy or immunotherapy to increase the responsiveness of the malignant cells to subsequent chemotherapy or immunotherapy or to speed the recovery of the bone marrow through induction of restorative cytokines such as GM-CSF. CpG nucleic acids also increase natural killer cell lytic activity and antibody dependent cellular cytotoxicity (ADCC). Induction of NK activity and ADCC may likewise be beneficial in cancer immunotherapy, alone or in conjunction with other treatments.

Another use of the described immunostimulatory nucleic acid molecules is in desensitization therapy for allergies, which are generally caused by IgE antibody generation against harmless allergens. The cytokines that are induced by unmethylated CpG nucleic acids are predominantly of a class called "Th1" which is most marked ;by a cellular immune response and is associated with Il-12 and IFN-.gamma.. The other major type of immune response is termed a Th2 immune response, which is associated with more of an antibody immune response and with the production of IL-4, Il-5 and IL-10. In general, it appears that allergic diseases are mediated by Th2 type immune responses and autoimmune diseases by Th1 immune response. Based on the ability of the immunostimulatory nucleic acid molecules to shift the immune response in a subject from a Th2 (which is associated with production of IgE antibodies and allergy) to a Th1 response (which is protective against allergic reactions), an effective dose of an immunostimulatory nucleic acid (or a vector containing a nucleic acid) alone or in conjunction with an allergen can be administered to a subject to treat or prevent an allergy.

Nucleic acids containing unmethylated CpG motifs may also have significant therapeutic utility in the treatment of asthma. Th2 cytokines, especially IL-4 and IL-5 are 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-.gamma. and IL-12, can suppress the formation of Th2 clones and production of Th2 cytokines.

As described in detail in the following Example 12, oligonucleotides containing an unmethylated CpG motif (I, e,. TCCATGACGTTCCTGACGTT; SEQ ID NO. 10) but not a control oligonucleotide (TCCATGAGCTTCCTGAGTCT; SEQ ID NO. 8) prevented the development of an inflammatory cellular infiltrate and eosinophilia in a murine model of asthma. Furthermore, the suppression of eosinophilic inflammation was associated with a suppression of a Th2 response and induction of a Th1 response.

For use in therapy, an effective amount of an appropriate immunostimulatory nucleic acid molecule alone or formulated as a delivery complex can be administered to a subject by any mode allowing the oligonucleotide to be taken up by the appropriate target cells (e.g., B-cells and monocytic cells). Preferred routes of administration include oral and transdermal (e.g., via a patch). Examples of other routes of administration include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.). The injection can be in a bolus or a continuous infusion.

A nucleic acid alone or as a nucleic acid delivery complex can be administered in conjunction with a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" is intended to include substances that can be coadministered with a nucleic acid or a nucleic acid delivery complex and allows the nucleic acid to perform its indicated function. Examples of such carriers include solutions, solvents, dispersion media, delay agents, emulsions and the like. The use of such media for pharmaceutically active substances are well known in the art. Any other conventional carrier suitable for use with the nucleic acids falls within the scope of the instant invention.

The term "effective amount" of a nucleic acid molecule refers to the amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of a nucleic acid containing at least one unmethylated CpG for treating an immune system deficiency could be that amount necessary to eliminate a tumor, cancer, or bacterial, viral or fungal infection. An effective amount for use as a vaccine adjuvant could be the amount useful for boosting a subjects immune response to a vaccine. An "effective amount" for treating asthma can be that amount; useful for redirecting a Th2 type of immune response that is associated with asthma to a Th1 type of response. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular nucleic acid being administered (e.g. the number of unmethylated CpG motifs or their location in the nucleic acid), the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular oligonucleotide without necessitating undue experimentation.

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

EXAMPLES

Example 1

Effects of ODNs on B Cell Total RNA Synthesis and Cell Cycle

B cells were purified from spleens obtained from 6-12 week old specific pathogen free DBA/2 or BXSB mice (bred in the University of Iowa animal care facility; no substantial strain differences were noted) that were depleted of T cells with anti-Thy-1.2 and complement and centrifugation over lymphocyte M(Cedarlane Laboratories, Homby, Ontario, Canada) ("B cells"). B cells contained fewer than 1% CD4.sup.+ or CD8.sup.+ cells. 8.times.10.sup.4B cells were dispensed in triplicate into 96 well microtiter plates in 100 .mu.l RPMI containing 10% FBS (heat inactivated to 65.degree. C. for 30 min.), 50 .mu.M 2-mercaptoethanol, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2 mM L-glutamate. 20 .mu.M ODN were added at the start of culture for 20 h at 37.degree. C., cells pulsed with 1 .mu.Ci of .sup.3H uridine, and harvested and counted 4 hr later. Ig secreting B cells were enumerated using the ALISA spot assay after culture of whole spleen cells with ODN at 20 .mu.M for 48 hr. Data, reported in Table 1, represents the stimulation index compared to cell cultured without ODN. .sup.3H thymidine incorporation assays: showed similar results, but with some nonspecific inhibition by thymidine released from degraded ODN (Matson. S and A. M. Krieg (1992) Nonspecific suppression of .sup.3H-thymidine incorporation by control oligonucleotides. Antisense Research and Development 2:325).

Example 2

Effects of ODN on Production of IgM from B Cells

Single cell suspensions form the spleens of freshly killed mice were treated with anti-Thyl, anti-CD4, and anti-CD8 and complement by the method of Leibson et al., J. Exp. Med. 154:1681 (1981)). Resting B cells (<02% T cell contamination) were isolated from the 63-70% band of a discontinuous Percoll gradient by the procedure of DeFranco et al, J. Exp. Med. 155:1523 (1982). These were cultured as described above in 30 .mu.g/ml LPS for 48 hr. The number of B cells actively secreting IgM was maximal at this time point, as determined by ELIspot assay (Klinman, D. M. et al. J. Immunol 144:506 (1990)). In that assay, B cells were incubated for 6 hrs on anti-Ig coated microtiter plates. The Ig they produced (>99% IgM) was detected using phosphatase-labeled anti-Ig (Southern Biotechnology Associated, Birmingham, Ala.). The antibodies produced by individual B cells were visualized by addition of BCIP (Sigma Chemical Co., St. Louis Mo.) which forms an insoluble blue precipitate in the presence of phosphatase. The dilution of cells producing 20-40 spots/well was used to determine the total number of antibody-secreting B cells/sample. All assays were performed in triplicate (data reported in Table 1). In some experiments, culture supernatants were assayed for IgM by ELISA, and showed similar increased in response to CpG-ODN.

Example 3

B cell Stimulation by Bacterial DNA

DBA/2 B cells were cultured with no DNA or 50 .mu.g/ml of a (Micrococcus lysodeikticus; b) NZB/N mouse spleen; and c) NSF/N mouse spleen genomic DNAs for 48 hours, then pulsed with .sup.3H thymidine for 4 hours prior to cell harvest. Duplicate DNA samples were digested with DNASE I for 30 minutes at 37 C prior to addition to cell cultures. E coli DNA also induced an 8.8 fold increase in the number of IgM secreting B cells by 48 hours using the ELISAspot assay.

DBA/2 B cells were cultured with either no additive, 50 .mu.g/ml LPS or the ODN 1; 1a; 4; or 4a at 20 .mu.M. Cells were cultured and harvested at 4, 8, 24 and 48 hours. BXSB cells were cultured as in Example 1 with 5, 10, 20, 40 or 80 .mu.M of ODN 1; 1a; 4; or 4a or LPS. In this experiment, wells with no ODN had 3833 cpm. Each experiment was performed at least three times with similar results. Standard deviations of the triplicate wells were <5%.

Example 4

Effects of ODN on Natural Killer (NK) Activity

10.times.10.sup.6 C57BL/6 spleen cells were cultured in two ml RPMI (supplemented as described for Example 1) with or without 40 .mu.M CpG or non-CpG ODN for forty-eight hours. Cells were washed, and then used as effector cells in a short term .sup.51Cr release assay with YAC-1 and 2C11, two NK sensitive target cell lines (Ballas, Z. K. et al (1993) j. IMMUNOL. 150:17). Effector cells were added at various concentrations to 10.sup.4 51Cr-labeled target cells in V-bottom microtiter plates in 0.2 ml, and incubated in 5% CO.sub.2 for 4 hr. At 37.degree. C. Plates were then centrifuged, and an aliquot of the supernatant counted for radioactivity. Percent specific lysis was determined by calculating the ratio of the .sup.51Cr released in the presence of effector cells minus the .sup.51Cr released when the target cells are cultured alone, over the total counts released after cell lysis in 2% acetic acid minus the .sup.51Cr cpm released when the cells are cultured alone.

Example 5

In vivo Studies with CpG Phosphorothioate ODN

Mice were weighted and injected IP with 0.25 ml of sterile PBS or the indicated phosphorothioate ODN dissolved in PBS. Twenty four hours later, spleen cells were harvested, washed, and stained for flow cytometry using phycoerythrin conjugated 6B2 to gate on B cells in conjunction with biotin conjugated anti Ly-6A/E or anti-Ia.sup.d (Pharmingen, San Diego, Calif.) or anti-Bla-1 (Hardy, R. R. et al., J. Exp. Med 159:1169 (1984). Two mice were studied for each condition and analyzed individually.

Example 6

Titration of Phosphorothioate ODN for B Cell Stimulation

B cells were cultured with phosphorothioate ODN with the sequence of control ODN 1a or the CpG ODN 1d and 3Db and then either pulsed after 20 hr with .sup.3H uridine or after 44 hr with .sup.3H thymidine before harvesting and determining cpm.

Example 7

Rescue of B Cells From Apoptosis

WEHI-231 cells (5.times.10.sup.4/well) were cultured for 1 hr. At 37 C in the presence or absence of LPS or the control ODN 1a or the CpG ODN 1d and 3Db before addition of anti-IgM (1 .mu./ml). Cells were cultured for a further 20 hr. Before a 4 hr. Pulse with 2 .mu.Ci/well .sup.3H thymidine. In this experiment, cells with no ODN or anti-IgM gave 90.4.times.10.sup.3 cpm of .sup.3H thymidine incorporation by addition of anti-IgM. The phosphodiester ODN shown in Table 1 gave similar protection, though some nonspecific suppression due to ODN degradation. Each experiment was repeated at least 3 times with similar results.

Example 8

In vivo Induction of Murine IL-6

DBA/2 female mice (2 mos. old) were injected IP with 500 g CpG or control phosphorothioate ODN. At various time points after injection, the mice were bled. Two mice were studied for each time point. IL-6 was measured by Elisa, and IL-6 concentration was calculated by comparison to a standard curve generate using recombinant IL-6. The sensitivity of the assay was 10 pg/ml. Levels were undetectable after 8 hrs.

Example 9

Systemic Induction of Murine IL-6 Transcription

Mice and cell lines. DBA/2, BALB/c, and C3H/HeJ mice at 5-10 wk of age were used as a source of lymphocytes. All mice were obtained from The Jackson Laboratory (Bar Harbor, Me.), and bred and maintained under specific pathogen-free conditions in the University of Iowa Animal Care Unit. The mouse B cell line CH12.LX was kindly provided by Dr. G. Bishop (University of Iowa, Iowa City).

Cell preparation. Mice were killed by cervical dislocation. Single cell suspensions were prepared aseptically from the spleens from mice. T cell depleted mouse splenocytes were prepared by using anti-Thy-1.2 and complement and centrifugation over lymphocyte M (Cedarlane Laboratories, Homby, Ontario, Canada) as described (Krieg, A. M. et al., (1989) A role for endogenous retroviral sequences in the regulation of lymphocyte activation. J. Immunol 143:2448).

ODN and DNA. Phosphodiester oligonucleotides (O-ODN) and the backbone modified phosphorothioate oligonucleotides (S-ODN) were obtained from the DNA Core facility at the University of Iowa or from Operon Technologies (Alameda, Calif.). E. Coli DNA (Strain B) and calf thymus DNA were purchased from Sigma (St. Louis, Mo.). All DNA and ODN were purified by extraction with phenol:chlotoform:isoamyl alcohol (25:24:1) and/or ethanol precipitation. E. Coli and calf thymus DNA were single stranded prior to use by boiling for 10 min. followed by cooling on ice for 5 min. For some experiments, E. Coli and calf thymus DNA were digested with DNase 1(2 U/.mu.g of DNA) at 37.degree. C. for 2 hr in 1.times.SSC with 5 mM MgC12. To methylate the cytosine in CpG dinucleotide in E. Coli DNA, E. Coli DNA was treated with CpG methylase (M. SssI; 2 U/.mu.g of DNA) in NEBuffer 2 supplemented with 160 .mu.M S-adenosyl methionine and incubated overnight at 37.degree. C. Methylated DNA was purified as above. Efficiency of methylation was confirmed by Hpa II digestion followed by analysis by gel electrophoresis. All enzymes were purchased from New England Biolabs (Beverly, Mass.). LPS level in ODN was less than 12.5 ng/mg and E. Coli and calf thymus DNA contained less than 2.5 ng of LPS/mg of DNA by Limulus assay.

Cell Culture. All cells were cultured at 37.degree. C. in a 5% CO.sub.2 humidifier incubator maintained in RPMI-1640 supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS), 1.5 mM L-glutamine, 50 .mu.g/ml), CpG or non-CpG phosphodiester ODN (O0ODN) (20 .mu.M), phosphorothioate ODN (S-ODN) (0.5 .mu.M), or E. coli or calf thymus DNA (50 .mu.g/ml) at 37.degree. C. for 24 hr. (for IL-6 production) or 5 days ( for IgM production). Concentrations of stimulants were chosen based on preliminary studies with titrations. In some cases, cells were treated with CpG O-ODN along with various concentrations (1-10 .mu.g/ml) of neutralizing rat IgG1 antibody against murine IL-6 (hybridoma MP5-20F3) or control rate IgG1 mAB to E. Coli b-galactosidase (hybridoma GL 113; ATCC, Manassas, Va.) (20) for 5 days. At the end of incubation, culture supernatant fractions were analyzed by ELISA as below.

In vivo induction of IL-6 and IgM. BALB/c mice were injected intravenously (iv) with PBS, calf thymus DNA (200 .mu.g/100 .mu.l PBS/mouse), E. coli DNA (200 .mu.g/100 .mu.l PBS/mouse), or CpG or non-CpG S-ODN (200 .mu.g/100 .mu.l PBS/mouse). Mice (two/group) were bled by retroorbital puncture and sacrificed by cervical dislocation at various time points. Liver, spleen, thymus, and bone marrow were removed by RNA was prepared from those organs using RNAzol B (Tel-Test, Friendswood, Tex.) according to the manufactures protocol.

ELISA. Flat-bottomed Immun 1 plates (Dynatech Laboratories, Inc., Chantilly, Va.) were coated with 100 .mu.l/well of anti-mouse IL-6 mAb (MP5-20F3) (2 .mu.g/ml) or anti-mouse IgM .mu.-chain specific (5 .mu.g/ml; Sigma, St. Louis, Mo.) in carbonate-bicarbonate, pH 9.6 buffer (15 nM Na.sub.2CO.sub.3, 35 mM NaHCO.sub.3) overnight at 4.degree. C. The plates were then washed with TPBS (0.5 mM MgCl.sub.206H.sub.20, 2.68 mM KC1, 1.47 mM KH.sub.2PO.sub.4, 0.14 M NaCl, 6.6 mM K.sub.2HPO.sub.4, 0.5% Tween 20)and blocked with 10% FCS and TPBS for 2 hr at room temperature and then washed again. Culture supernatants, mouse sera, recombinant mouse IL-6 (Pharmigen, San Diego, Calif.) or purified mouse IgM (Calbiochem, San Diego, Calif.) were appropriately diluted in 10% FCS and incubated in triplicate wells for 6 hr at room temperature. The plates were washed and 100 .mu.l/well of biotinylated rat anti-mouse IL-6 monoclonal antibodies (MP5-32C11, Pharmingen, San Diego, Calif.) (1 .mu.g/ml in 10% FCS) or biotinylated anti-mouse Ig (Sigma, St. Louis, Mo.) were added and incubated for 45 min. at room temperature following washes with TPBS. Horseradish peroxidase (HRP) conjugated avidin (Bio-rad Laboratories, Hercules, Calif.) at 1:4000 dilution in 10% FCS (100 .mu.l/well) was added and incubated at room temperature for 30 min. The plates were washed and developed with o-phenylenediamine dihydrochloride (OPD; Sigma, St. Louis Mo.) 0.05 M phosphate-citrate buffer, pH 5.0, for 30 min. The reaction was stopped with 0.67 N H.sub.2SO.sub.4 and plates were read on a microplate reader (Cambridge Technology, Inc., Watertown, Mass.) at 490-600 nm. The results are shown in FIGS. 1 and 2.

RT-PCR A sense primer, an antisense primer, and an internal oligonucleotide probe for IL-6 were synthesized unpublished sequences (Montgomery, R. A. and M. S. Dallman (1991), Analysis for cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction (J. Immunol.) 147:554). cDNA synthesis and IL-6 PCR was done essentially as described by Montgomery and Dallman (Montgomery, R. A. And M. S. Dallman (1991), Analysis of cytokine gene expression during fetal thymic ontogeny using the polymerase chain reaction (J. Immunol.) 147:554) using RT-PCR reagents from Perkin-Elmer Corp. (Hayward, Calif.). Samples were analyzed after 30 cycles of amplification by gel electrophoresis followed by unblot analysis (stoye, J. P. et al., (1991) DNA hybridization in dried gels with fragmented probes: an improvement over blotting techniques, Techniques 3:123). Briefly, the gel was hybridized at room temperature for 30 min. in denaturation buffer (0.05 M Na0H, 1.5 M NaCl) followed by incubation for 30 min. In renaturation buffer (1.5 M NaCl, 1 M Tris, pH 8) and a 30 min. Wash in double distilled water. The gel was dried and prehybridized at 47.degree. C. for 2 hr. Hybridization buffer (5.times.SSPE, 0.1% SDS) containing 10 .mu.g/ml denatured salmon sperm DNA. The gel was hybridized with 2.times.10.sup.6 cpm/ml g-[.sup.32 P] ATP end-labeled internal oligonucleotide probe for IL-6 (5'CATTTCCACGATTTCCCA3') SEQ ID. NO: 118) overnight at 47.degree. C., washed 4 times (2.times.SSC, 0.2% SDS) at room temperature and autoradiographed. The results are shown in FIG. 3.

Cell Proliferation assay. DBA/2 mice spleen B cells (5.times.10.sup.4 cells/100 .mu.l/well) were treated with media, CpG or non-CpG S-ODN (0.5 .mu.M) or O-ODN (20 .mu.M) for 24 hr at 37.degree. C. Cells were pulsed for the last four hr. With either [.sup.3H] Thymidine or [.sup.3H] Uridine (1 .mu.Ci/well). Amounts of [.sup.3H] incorporated were measured using Liquid Scintillation Analyzer (Packard Instrument Co., Downers Grove, Ill.).

Transfections and Cat assays. WEHI-231 cells (10.sup.7 cells) were electroporated with 20 .mu.g of control or human IL-6 promoter-CAT construct (kindly provided by S. Manolagas, Univ. of Arkansas) (Pottratz, S. T. Et al., (1994) 17B-estradiol inhibits expression of human interleukin-6 promotor-reporter constructs by a receptor-dependent mechanism. J. Clin. Invest. 93:944) at 250 mV and 960 .mu.F. Cells were stimulated with various concentrations of CpG or non-CpG ODN after electroporation. Chloramphenicol acetyltransferase (CAT) activity was measured by a solution assay (Seed, B. and J. Y. Sheen (1988) A single phase-extraction assay for chloramphenicol acetyl transferase activity. Gene 76:271) 16 hr. after transfection. The results are presented in FIG. 5.

Example 10

Oligodeoxynucleotide Modifications Determine the Magnitude of B Cell Stimulation by CpG Motifs

ODN were synthesized on an Applied Biosystems Inc. (Foster City, Calif.) model 380A, 380B, or 394 DNA synthesizer using standard procedures (Beacage and Caruthers (1981) Deoxynucleoside phosphorarnidites--A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedon Letters 22, 1859-1862.). Phosphodiester ODN were synthesized using standard beta-cyanoethyl phosphoramidite chemistry. Phosphorothioate linkages were introduced by oxidizing the phosphite linkage with elemental sulfur instead of the standard iodine oxidation. The four common nucleoside phosphoramidites were purchased from Applied Biosystems. All phosphodiester and thioate containing ODN were protected by treatment with concentrated ammonia at 55.degree. C. for 12 hours. The ODN were purified by gel exclusion chromatography and lyophilized to dryness prior to use. Phosphorodithioate linkages were introduced by using deoxynucleoside S-(b-benzoylmercaptoethyl) pyrrolidino thiophosphoramidites (Wiesler, W. T. et al.,(1993) In Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs-Synthesis and Properties, Agrawal, S. (Ed.), Humana Press, 191-206.). Dithioate containing ODN were deprotected by treatment with concentrated ammonia at 55.degree. C. for 12 hours followed by reverse phase HPLC purification.

In order to synthesize oligomers containing methylphosphonothioates or methylphosphonates as well as phosphodiesters at any desired internucleotide linkage, two different synthetic cycles were used. The major synthetic differences in two cycles are the coupling reagent where dialkylarninomethylnucleoside phosphines are used and the oxidation reagents in the case of methylphosphonothioates. In order to synthesize either derivative, the condensation time has been increased for the dialkylaminomethylnucleoside phosphines due to the slower kinetics of coupling (Jager and Engels, (1984) Synthesis of deoxynucleoside methylphosphonates via a phosphonamidite approach. Tetrahedron Letters 24, 1437-1440). After the coupling step has been completed, the methylphosphnodiester is treated with the sulfurizing reagent (5% elemental sulfur, 100 millimolar N,N-diarnethylaminopyridine in carbon disulfide/pyridine/triethylamine), four consecutive times for 450 seconds each to produce methylphosphonothioates. To produce methylphosphonate linkages, the methylphosphinodiester is treated with standard oxidizing reagent (0.1 M iodine in tetrahydrofuran/2,6-lutidine/water).

The silica gel bound oligomer was treated with distilled pyridine/concentrated ammonia, 1:1, (v/v) for four days at 4 degrees centigrade. The supernatant was dried in vacuo, dissolved in water and chromatographed on a G50/50 Sephadex column.

As used herein, O-ODN refers to ODN which are phosphodiester; S-ODN are completely phosphorothioate modified; S-O=ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are phosphorothioate modified; 2.sub.2-O-ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are phosphorodithioate modified; and MP-O-ODN are chimeric ODN in which the central linkages are phosphodiester, but the two 5' and five 3' linkages are methylphosphonate modified. The ODN sequences studied (with CpG dinucleotides indicated by underlining) include:

TABLE-US-00020 3D (5' GAGAACGCTGGACCTTCCAT); (SEQ. ID. NO. 20) 3M (5' TCCATGTCGGTCCTGATGCT); (SEQ. ID. NO. 28) 5 (5' GGCGTTATTCCTGACTCGCC); (SEQ. ID. NO. 99) and 6 (5' CCTACGTTGTATGCGCCCAGCT). (SEQ. ID NO. 100)

These sequences are representative of literally hundreds of CpG and non-CpG ODN that have been tested in the course of these studies.

Mice. DBA/2, or BXSB mice obtained from The Jackson Laboratory (Bar Harbor, Me.), and maintained under specific pathogen-free conditions were used as a source of lymphocytes at 5-10 wk of age with essentially identical results.

Cell proliferation assay. For cell proliferation assays, mouse spleen cells (5.times.10.sup.4 cells/100 .mu.l/well) were cultured at 37.degree. C. in a 5% CO.sub.2 humidified incubator in RPMI-1640 supplemented with 10% (v/v) heat inactivated fetal calf serum (heated to 65.degree. C. for experiments with O-ODN, or 56.degree. C. for experiments using only modified ODN), 1.5 .mu.M L-glutamine, 50 .mu.M 2-mercaptoethanol, 100 U/ml penicillin and 100 .mu.g/ml streptomycin for 24 hr or 48 hr as indicated. 1 .mu.Ci of .sup.3H uridine or thymidine (as indicated) was added to each well, and the cells harvested after an additional 4 hours of culture. Filters were counted by scintillation counting. Standard deviations of the triplicate wells were <5%. The results are presented in FIGS. 6-8.

Example 11

Induction of NK Activity

Phosphodiester ODN were purchased form Operon Technologies (Alameda, Calif.). Phosphorothioate ODN were purchased from the DNA core facility, University of Iowa, or from The Midland Certified Reagent Company (Midland Tex.). E. coli (strain B) DNA and calf thymus DNA were purchased from Sigma (St. Louis, Mo.). All DNA and ODN were purified by extraction with phenol:chloroform:isoamyl alcohol (25:24:1) and/or ethanol precipitation. The LPS level in ODN was less than 12.5 ng/mg and E. coli and calf thymus DNA contained less than 2.5 ng of LPS/mg of DNA by Limulus assay.

Virus-free, 4-6 week old, DBA/2, C57BL/6 (B6) and congenitally thymic BALB/C mice were obtained on contract through the Veterans Affairs from the National Cancer Institute (Bethesda, Md.). C57BL/6 SCID mice were bred in the SPF barrier facility at the University of Iowa Animal Care Unit.

Human peripheral monucluclear blood leukocytes (PBMC) were obtained as previously described (Ballas, Z. K. et al., (1990) J. Allergy Clin. Immunol. 85:453; Ballas, Z. K. And W. Rasmussen (1990) J. Immunol. 145:1039; Ballas, Z. K. and W. Rasmussen (1993) J. Immunol. 150;17). Human or murine cells were cultured at 5.times.10.sup.6/well, at 37.degree. C. in a 5% CO.sub.2 humidified atmosphere in 24-well plates (Ballas, Z. K. Et al., (1990) J. Allergy Clin. Immunol. 85:453; Ballas, Z. K. And W. Rasmussen (1990) J. Immunol 145:1039; and Ballas, Z. K. and W. Rasmussen (1193) J. Immunol, 150:17), with medium alone or with CpG or non-CpG ODN at the indicated concentrations, or with E. coli or calf thymus (50 .mu.g/ml) at 37.degree. C. for 24 hr. All cultures were harvested at 18 hr. and the cells were used as effectors in a standard 4 hr. .sup.51Cr-release assay against K562 (human) or YAC-1 (mouse) target cells as previously described. For calculation of lytic units (LU), 1 LU was defined as the number of cells needed to effect 30% specific lysis. Where indicated, neutralizing antibodies against IFN-.beta. (Lee Biomolecular, San Diego, Calif.) or IL-12 (C15.1, C15.6, C17.8, and C17.15; provided by Dr. Giorgio Trinchieri, The Winstar Institute, Philadelphia, Pa.) or their isotype controls were added at the initiation of cultures to a concentration of 10 .mu.g/ml. For anti-IL-12 additional, 10 .mu.g of each of the 4 MAB (or isotype controls) were added simultaneously. Recombinant human IL-2 was used at a concentration of 100 U/ml.

Example 12

Prevention of the Development of an Inflammatory Cellular Infiltrate and Eosinophilia in a Murine Model of Asthma

6-8 week old C56BL/6 mice (from The Jackson Laboratory, Bar Harbor, Me.) were immunized with 5,000 Schistosoma mansoni eggs by intraperitoneal (i.p.) injection on days 0 and 7. Schistosoma mansoni eggs contain an antigen (Schistosoma mansoni egg antigen (SEA)) that induces a Th2 immune response (e.g. production of IgE antibody). IgE antibody production is known to be an important cause of asthma.

The immunized mice were then treated with oligonucleotides (30 .mu.g in 200 .mu.l saline by i.p. injection), which either contained an unmethylated CpG motif (i.e., TCCATGACGTTCCTGACGTT; SEQ ID NO.10) or did the (i.e., control, TCCATGAGCTTCCTGAGTCT; SEQ ID NO.8). Soluble SeEA (10 .mu.g in 25 .mu.l of saline) was administered by intranasal instillation on days 14 and 21. Saline was used as a control.

Mice were sacrificed at various times after airway challenge. Whole lung lavage was performed to harvest airway and alveolar inflammatory cells. Cytokine levels were measured from lavage fluid by ELISA. RNA was isolated from whole lung for Northern analysis and RT-PCR studies using CsCl gradients. Lungs were inflated and perfused with 4% paraformaldehyde for histologic examination.

FIG. 9 shows that when the mice are initially injected with the eggs i.p., and then inhale the egg antigen (open circle), many inflammatory cells are present in the lungs. However, when the mice are initially given a nucleic acid containing an unmethylated CpG motif along with the eggs, the inflammatory cells in the lung are not increased by subsequent inhalation of the egg antigen (open triangles).

FIG. 10 shows that the same results are obtained only when eosinophils present in the lung lavage are measured. Eosinophils are the type of inflammatory cell most closely associated with asthma.

FIG. 11 shows that when the mice are treated with a control oligo at the time of the initial exposure to the egg, there is little effect on the subsequent influx of eosinophils into the lungs after inhalation of SEA. Thus, when mice inhale the eggs on days 14 or 21, they develop an acute inflammatory response in the lungs. However, giving a CpG oligo along with the eggs at the time of initial antigen exposure on days 0 and 7 almost completely abolishes the increase in eosinophils when the mice inhale the egg antigen on day 14.

FIG. 12 shows that very low doses of oligonucleotide (<10 .mu.g) can give this protection.

FIG. 13 shows that the resultant inflammatory response correlates with the levels of the Th2 cytokine IL-4 in the lung.

FIG. 14 shows that administration of an oligonucleotide containing an unmethylated CpG motif can actually redirect the cytokine response of the lung to production of Il-12, indicating the Th1 type of immune response.

FIG. 15 shows that administration of an oligonucleotide containing an uuniethylated CpG motif can also redirect the cytokine response of the lung to production of IFN-.gamma., indicating a Th1 type of immune response.

Example 13

CpG Oligonucleotides Induce Human PBMC to Secrete Cytokines

Human PBMC were prepared from whole blood by standard centrifugation over Ficoll hypaque. Cells (5.times.10.sup.5/ml) were cultured in 10% autologous serum in 95 well microtiter plates with CpG or control oligodeoxynucleotides (24 .mu.g/ml for phosphodiester oligonucleotides; 6 g/ml for nuclease resistant phosphorothioate oligonucleotides) for 4 hr in the case of TNF-.alpha. or 24 hr. For the other cytokines before supernatant harvest and assay, measured by ELISA using Quantikine kits or reagents from R&D Systems (pg/ml) or cytokine ELISA kits from Biosource (for IL-12 assay). Assays were performed as per the manufacturer's instructions. Data are presented in Table 6 as the level of cytokine above that in wells with no added oligodeoxynucleotide.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

SEQUENCE LISTINGS

1

123 1 20 DNA Artificial Sequence Synthetic Oligonucleotide 1 atggaaggtc cagcgttctc 20 2 20 DNA Artificial Sequence Synthetic Oligonucleotide 2 atcgacctac gtgcgttctc 20 3 20 DNA Artificial Sequence Synthetic Oligonucleotide 3 tccataacgt tcctgatgct 20 4 15 DNA Artificial Sequence Synthetic Oligonucleotide 4 gctagatgtt agcgt 15 5 19 DNA Artificial Sequence Synthetic Oligonucleotide 5 gagaacgtcg accttcgat 19 6 15 DNA Artificial Sequence Synthetic Oligonucleotide 6 gcatgacgtt gagct 15 7 20 DNA Artificial Sequence Synthetic Oligonucleotide 7 tccatgacgt tcctgatgct 20 8 20 DNA Artificial Sequence Synthetic Oligonucleotide 8 tccatgagct tcctgagtct 20 9 20 DNA Artificial Sequence Synthetic Oligonucleotide 9 tccaagacgt tcctgatgct 20 10 20 DNA Artificial Sequence Synthetic Oligonucleotide 10 tccatgacgt tcctgacgtt 20 11 21 DNA Artificial Sequence Synthetic Oligonucleotide 11 tccatgagct tcctgagtgc t 21 12 20 DNA Artificial Sequence Synthetic Oligonucleotide 12 ggggtcaacg ttgagggggg 20 13 15 DNA Artificial Sequence Synthetic Oligonucleotide 13 gctagangtt agcgt 15 14 15 DNA Artificial Sequence Synthetic Oligonucleotide 14 gctagacgtt agngt 15 15 20 DNA Artificial Sequence Synthetic Oligonucleotide 15 atcgactctc gagcgttctc 20 16 20 DNA Artificial Sequence Synthetic Oligonucleotide 16 atngactctn gagngttctc 20 17 20 DNA Artificial Sequence Synthetic Oligonucleotide 17 atngactctc gagcgttctc 20 18 20 DNA Artificial Sequence Synthetic Oligonucleotide 18 atcgactctc gagcgttntc 20 19 20 DNA Artificial Sequence Synthetic Oligonucleotide 19 atggaaggtc caacgttctc 20 20 20 DNA Artificial Sequence Synthetic Oligonucleotide 20 gagaacgctg gaccttccat 20 21 20 DNA Artificial Sequence Synthetic Oligonucleotide 21 gagaacgctc gaccttccat 20 22 20 DNA Artificial Sequence Synthetic Oligonucleotide 22 gagaacgctc gaccttcgat 20 23 20 DNA Artificial Sequence Synthetic Oligonucleotide 23 gagcaagctg gaccttccat 20 24 20 DNA Artificial Sequence Synthetic Oligonucleotide 24 gagaangctg gaccttccat 20 25 20 DNA Artificial Sequence Synthetic Oligonucleotide 25 gagaacgctg gacnttccat 20 26 20 DNA Artificial Sequence Synthetic Oligonucleotide 26 gagaacgatg gaccttccat 20 27 20 DNA Artificial Sequence Synthetic Oligonucleotide 27 gagaacgctc cagcactgat 20 28 20 DNA Artificial Sequence Synthetic Oligonucleotide 28 tccatgtcgg tcctgatgct 20 29 20 DNA Artificial Sequence Synthetic Oligonucleotide 29 tccatgctgg tcctgatgct 20 30 20 DNA Artificial Sequence Synthetic Oligonucleotide 30 tccatgtngg tcctgatgct 20 31 20 DNA Artificial Sequence Synthetic Oligonucleotide 31 tccatgtcgg tnctgatgct 20 32 20 DNA Artificial Sequence Synthetic Oligonucleotide 32 tccatgtcgg tcctgctgat 20 33 20 DNA Artificial Sequence Synthetic Oligonucleotide 33 tccatgccgg tcctgatgct 20 34 20 DNA Artificial Sequence Synthetic Oligonucleotide 34 tccatggcgg tcctgatgct 20 35 20 DNA Artificial Sequence Synthetic Oligonucleotide 35 tccatgacgg tcctgatgct 20 36 20 DNA Artificial Sequence Synthetic Oligonucleotide 36 tccatgtcga tcctgatgct 20 37 20 DNA Artificial Sequence Synthetic Oligonucleotide 37 tccatgtcgc tcctgatgct 20 38 20 DNA Artificial Sequence Synthetic Oligonucleotide 38 tccatgtcgt tcctgatgct 20 39 20 DNA Artificial Sequence Synthetic Oligonucleotide 39 tccatgacgt ccctgatgct 20 40 20 DNA Artificial Sequence Synthetic Oligonucleotide 40 tccatcacgt gcctgatgct 20 41 19 DNA Artificial Sequence Synthetic Oligonucleotide 41 ggggtcagtc ttgacgggg 19 42 15 DNA Artificial Sequence Synthetic Oligonucleotide 42 gctagacgtt agtgt 15 43 15 DNA Artificial Sequence Synthetic Oligonucleotide 43 gctagacntt agtgt 15 44 20 DNA Artificial Sequence Synthetic Oligonucleotide 44 tccatgtngt tcctgatgct 20 45 18 DNA Artificial Sequence Synthetic Oligonucleotide 45 tctcccagcg tgcgccat 18 46 24 DNA Artificial Sequence Synthetic Oligonucleotide 46 tcgtcgtttt gtcgttttgt cgtt 24 47 20 DNA Artificial Sequence Synthetic Oligonucleotide 47 tcgtcgttgt cgttgtcgtt 20 48 21 DNA Artificial Sequence Synthetic Oligonucleotide 48 tgtcgtttgt cgtttgtcgt t 21 49 22 DNA Artificial Sequence Synthetic Oligonucleotide 49 tcgtcgttgt cgttttgtcg tt 22 50 19 DNA Artificial Sequence Synthetic Oligonucleotide 50 tgtcgttgtc gttgtcgtt 19 51 14 DNA Artificial Sequence Synthetic Oligonucleotide 51 tcgtcgtcgt cgtt 14 52 20 DNA Artificial Sequence Synthetic Oligonucleotide 52 tcctgtcgtt ccttgtcgtt 20 53 20 DNA Artificial Sequence Synthetic Oligonucleotide 53 tcctgtcgtt ttttgtcgtt 20 54 21 DNA Artificial Sequence Synthetic Oligonucleotide 54 tcgtcgctgt ctgcccttct t 21 55 21 DNA Artificial Sequence Synthetic Oligonucleotide 55 tcgtcgctgt tgtcgtttct t 21 56 21 DNA Artificial Sequence Synthetic Oligonucleotide 56 gcgtgcgttg tcgttgtcgt t 21 57 6 DNA Artificial Sequence Synthetic Oligonucleotide 57 gtcgtt 6 58 6 DNA Artificial Sequence Synthetic Oligonucleotide 58 gtcgct 6 59 24 DNA Artificial Sequence Synthetic Oligonucleotide 59 accatggacg atctgtttcc cctc 24 60 18 DNA Artificial Sequence Synthetic Oligonucleotide 60 taccgcgtgc gaccctct 18 61 24 DNA Artificial Sequence Synthetic Oligonucleotide 61 accatggacg aactgtttcc cctc 24 62 24 DNA Artificial Sequence Synthetic Oligonucleotide 62 accatggacg agctgtttcc cctc 24 63 24 DNA Artificial Sequence Synthetic Oligonucleotide 63 accatggacg acctgtttcc cctc 24 64 24 DNA Artificial Sequence Synthetic Oligonucleotide 64 accatggacg tactgtttcc cctc 24 65 24 DNA Artificial Sequence Synthetic Oligonucleotide 65 accatggacg gtctgtttcc cctc 24 66 24 DNA Artificial Sequence Synthetic Oligonucleotide 66 accatggacg ttctgtttcc cctc 24 67 15 DNA Artificial Sequence Synthetic Oligonucleotide 67 cacgttgagg ggcat 15 68 15 DNA Artificial Sequence Synthetic Oligonucleotide 68 ctgctgagac tggag 15 69 12 DNA Artificial Sequence Synthetic Oligonucleotide 69 tcagcgtgcg cc 12 70 17 DNA Artificial Sequence Synthetic Oligonucleotide 70 atgacgttcc tgacgtt 17 71 17 DNA Artificial Sequence Synthetic Oligonucleotide 71 tctcccagcg ggcgcat 17 72 18 DNA Artificial Sequence Synthetic Oligonucleotide 72 tctcccagcg cgcgccat 18 73 20 DNA Artificial Sequence Synthetic Oligonucleotide 73 tccatgtcgt tcctgtcgtt 20 74 20 DNA Artificial Sequence Synthetic Oligonucleotide 74 tccatagcgt tcctagcgtt 20 75 21 DNA Artificial Sequence Synthetic Oligonucleotide 75 tcgtcgctgt ctccgcttct t 21 76 19 DNA Artificial Sequence Synthetic Oligonucleotide 76 tcctgacgtt cctgacgtt 19 77 19 DNA Artificial Sequence Synthetic Oligonucleotide 77 tcctgtcgtt cctgtcgtt 19 78 20 DNA Artificial Sequence Synthetic Oligonucleotide 78 tccatgtcgt ttttgtcgtt 20 79 20 DNA Artificial Sequence Synthetic Oligonucleotide 79 tccaggactt ctctcaggtt 20 80 20 DNA Artificial Sequence Synthetic Oligonucleotide 80 tccatgcgtg cgtgcgtttt 20 81 20 DNA Artificial Sequence Synthetic Oligonucleotide 81 tccatgcgtt gcgttgcgtt 20 82 20 DNA Artificial Sequence Synthetic Oligonucleotide 82 tccacgacgt tttcgacgtt 20 83 20 DNA Artificial Sequence Synthetic Oligonucleotide 83 gcggcgggcg gcgcgcgccc 20 84 25 DNA Artificial Sequence Synthetic Oligonucleotide 84 tgtcgttgtc gttgtcgttg tcgtt 25 85 13 DNA Artificial Sequence Synthetic Oligonucleotide 85 tgtcgttgtc gtt 13 86 20 DNA Artificial Sequence Synthetic Oligonucleotide 86 tccacgacgt tttcgacgtt 20 87 20 DNA Artificial Sequence Synthetic Oligonucleotide 87 tccatgacga tcctgatgct 20 88 20 DNA Artificial Sequence Synthetic Oligonucleotide 88 tccatgacgc tcctgatgct 20 89 15 DNA Artificial Sequence Synthetic Oligonucleotide 89 gctagacgtt agcgt 15 90 8 DNA Artificial Sequence Synthetic Oligonucleotide 90 tcaacgtt 8 91 8 DNA Artificial Sequence Synthetic Oligonucleotide 91 tcaagctt 8 92 8 DNA Artificial Sequence Synthetic Oligonucleotide 92 tcagcgct 8 93 8 DNA Artificial Sequence Synthetic Oligonucleotide 93 tcatcgat 8 94 8 DNA Artificial Sequence Synthetic Oligonucleotide 94 tcttcgaa 8 95 8 DNA Artificial Sequence Synthetic Oligonucleotide 95 ccaacgtt 8 96 8 DNA Artificial Sequence Synthetic Oligonucleotide 96 tcaacgtc 8 97 20 DNA Artificial Sequence Synthetic Oligonucleotide 97 tccaggactt tcctcaggtt 20 98 20 DNA Artificial Sequence Synthetic Oligonucleotide 98 ttcaggactt tcctcaggtt 20 99 20 DNA Artificial Sequence Synthetic Oligonucleotide 99 ggcgttattc ctgactcgcc 20 100 22 DNA Artificial Sequence Synthetic Oligonucleotide 100 cctacgttgt atgcgcccag ct 22 101 7 DNA Artificial Sequence Synthetic Oligonucleotide 101 tgtcgct 7 102 7 DNA Artificial Sequence Synthetic Oligonucleotide 102 tgtcgtt 7 103 7 DNA Artificial Sequence Synthetic Oligonucleotide 103 tgacgtc 7 104 8 DNA Artificial Sequence Synthetic Oligonucleotide 104 tgacgtca 8 105 6 DNA Artificial Sequence Synthetic Oligonucleotide 105 aacgtt 6 106 7 DNA Artificial Sequence Synthetic Oligonucleotide 106 caacgtt 7 107 8 DNA Artificial Sequence Synthetic Oligonucleotide 107 aacgttct 8 108 7 DNA Artificial Sequence Synthetic Oligonucleotide 108 tgacgtt 7 109 6 DNA Artificial Sequence Synthetic Oligonucleotide 109 gccggt 6 110 6 DNA Artificial Sequence Synthetic Oligonucleotide 110 gacggt 6 111 6 DNA Artificial Sequence Synthetic Oligonucleotide 111 gacgtc 6 112 6 DNA Artificial Sequence Synthetic Oligonucleotide 112 cacgtg 6 113 7 DNA Artificial Sequence Synthetic Oligonucleotide 113 cgacgtt 7 114 20 DNA Artificial Sequence Synthetic Oligonucleotide 114 atggaaggtc cagtgttctc 20 115 20 DNA Artificial Sequence Synthetic Oligonucleotide 115 atggactctc cagcgttctc 20 116 20 DNA Artificial Sequence Synthetic Oligonucleotide 116 atcgactctc gagngttctc 20 117 15 DNA Artificial Sequence Synthetic Oligonucleotide 117 gctagangtt agtgt 15 118 18 DNA Artificial Sequence Synthetic Oligonucleotide 118 catttccacg atttccca 18 119 21 DNA Artificial Sequence Synthetic Oligonucleotide 119 tcgtcgctgt ctgcccttct t 21 120 21 DNA Artificial Sequence Synthetic Oligonucleotide 120 tcgtcgctgt tgtcgtttct t 21 121 20 DNA Artificial Sequence Synthetic Oligonucleotide 121 tccttgtcgt tcctgtcgtt 20 122 20 DNA Artificial Sequence Synthetic Oligonucleotide 122 tccatgtngt tcctgtngtt 20 123 23 DNA Artificial Sequence Synthetic Oligonucleotide 123 tcgtcgtttt gtcgttttgt cgt 23

* * * * *

References


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