WO2006108358A1

WO2006108358A1 - ANTIVIRAL USE OF ARTIFICIAL CpG-CONTAINING SINGLE-STRANDED OLIGODEOXYNUCLEOTIDES IN COMBINATION WITH RIBAVIRIN

Info

Publication number: WO2006108358A1
Application number: PCT/CN2006/000667
Authority: WO
Inventors: Li-Ying Wang; Mu-Sheng Bao; Yong-Li Yu
Original assignee: Changchun Huapu Biotechnology Co., Ltd.
Priority date: 2005-04-13
Filing date: 2006-04-13
Publication date: 2006-10-19
Also published as: CN1847256B; CN1847256A

Abstract

The present invention provides a composition containing ribavirin and artificial CpG single-stranded oligodeoxynucleotides or a kit which can prevent or treat viral infection and infectious diseases caused by virus, and use thereof. The invention also provides a method for preventing or treating viral infection and infectious diseases caused by virus.

Description

Antiviral effect produced by the combined use of synthetic CpG-containing single-stranded deoxyoligonucleotides and ribavirin

The present invention relates to a synthetic CpG-containing single-stranded deoxynucleotide and ribavirin, in particular to a combination of artificially synthesized single-stranded deoxynucleotides and ribavirin, the artificial labor involved in the present invention The combined single-stranded deoxynucleotide and ribavirin can be used in the treatment and prevention of diseases associated with viral infections and viral infections. The viruses referred to in the present invention include, but are not limited to, influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and papillomavirus. The invention also provides sequences of synthetic single-stranded deoxynucleotides. Background of the invention

CpG ODN is a synthetic deoxyoligonucleotide single-stranded DNA containing one or more CpG dinucleotides, wherein CpG is a dinucleotide formed by cytosine and guanine linked by phosphate, and C represents a cell. Pyrimidine, G represents guanine, p represents phosphoric acid, and cytosine is located at the 5' end. CpG ODNs can take many forms due to differences in sequences, especially on both sides of CpG. Some CpG ODNs have clear immunomodulatory effects and show good clinical value (Weiner GJ. The immunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol 2000 Oct; 68(4): 455-63) Some CpG OD The effect is species-specific, that is, CpG ODN, which exhibits a biological effect on one animal, and does not necessarily exhibit the same nature or intensity of biological activity in another animal (Gunther Hartmann, et al. Delineation of a CpG Phosphorothioate) Oligodeoxynucleotide for Activating Primate Immune Responses In Vitro and In Vivol The Journal of Immunology, 2000, 164: 1617-1624).

According to the characteristics of the function, single-chain deoxynucleotides containing CpG are classified into three types: A-type single-stranded deoxynucleotides (CpG-A ODN), and B-type single-stranded deoxynucleotides (CpG-B ODN). And C-type single-stranded deoxynucleotides (CpG-C ODN). The single-stranded deoxynucleotides that act to activate dendritic cells, natural killer cells, and stimulate dendritic cells to secrete interferon activity are classified as type A single-stranded deoxynucleotides. Type A single-stranded deoxynucleotides are expected to be useful in the treatment of tumors, viral infectious diseases, and other diseases treated with interferon. The main manifestation is activation of B lymphocytes, and single-stranded deoxynucleotides that enhance humoral immune response activity are classified as type B single-stranded deoxynucleotides. Type B single-chain deoxynucleotides are a class of molecular adjuvants that enhance vaccine immunity. Effect. Single-stranded deoxynucleotides having both A-type single-stranded deoxynucleotides and B-type single-stranded deoxynucleotides active are classified as C-type single-stranded deoxynucleotides.

Experiments have shown that synthetic CpG-containing single-stranded deoxynucleotides have antiviral biological activity. Transvaginal synthesis of single-stranded deoxynucleotides can increase the ability of mice or guinea pigs to resist herpes simplex virus type 2 (HSV-2) (Richard B. Pyles et al. Journal of Virology, November 2002, Vol.76 , No. 22 p. 11387-11396). Administration of synthetic single-stranded deoxynucleotides to mice reduces the load of respiratory syncytial virus (RSV) (Cho JY, et al. J Allergy Clin Immunol 2001 Nov; 108(5): 697-702). In mice injected with synthetic single-stranded deoxynucleotides, viral titers in vaginal secretions were significantly reduced after a lethal dose of HSV-2 (Harandi AM, Eriksson K, Holmgren J. J Virol 2003 Jan; 77 ( 2): 953-62). Wo ¹ J ribavirin (l-6-D-ribofuranosyl -l, 2,4-triazole-3-carboxamide, Ribavirin), Virazole, also known, is a nucleoside analog with broad-spectrum antiviral activity ( Gilbert, BE et al. Agents Chemother. 1986, 30(2): 201-205) (Streeter DG, et al. Proc Natl Acad Sci US A. 1973 Apr; 70(4): 1174-8.). Treatment of a variety of viral infectious diseases (Gilbert, BE et al. Agents Chemother. 1986, 30(2): 201-205). The combination of ribavirin and alpha interferon is an effective method for the treatment of chronic hepatitis C caused by hepatitis C virus infection (Davis GL, et al. N Engl J Med. 1998 Nov 19;339(21): 1493 -9. ) . The combination of ribavirin and pegylated interferon (peginterferon) has a significant effect on the treatment of hepatitis C (Torriani FJ, et al. N Engl J Med. 2004 Jul 29; 351(5): 438-50 ). Influenza virus is the causative agent of human influenza. In 1918-1919, 20 million people worldwide died of the flu virus (Patterson, KD & Pyle, GF (1991) Bull. Hist. Med. 65, 4-212). Vaccination and the use of antiviral drugs are the two main methods of combating influenza viruses (Bridges, CB et al. (2001) Morbid. Mortal. Wkly. Rep. 50, 1-44), but it turns out that both methods The flu epidemic cannot be effectively controlled (Webby, RJ & Webster, RG (2001) Philos. Trans. R. Soc. London 356, 1817-1828). In susceptible populations, the short-term (six-month) protection rate for vaccination is approximately 39% (Fukuda, Κ., Ν. J. (1999) Morbid. Mortal. Wkly. Rep. 48, 1-28, Castle, SC Clin Infect Dis. 2000 Aug; 31(2): 578-85. Epub 2000 Sep 14. Review;). Due to the changing nature of influenza virus antigens [hemagglutinin (HA) and ceramidase (NA)], current vaccines have been remodeled almost every year. Due to the large side effects and the emergence of new anti-influenza strains, some approved anti-influenza drugs The effect is also not satisfactory (Luscher-Mattli, M. Arch Virol. 2000; 145(ll): 2333-48. Review.).

Hepatitis C virus (HCV) is the causative agent of hepatitis C and has infected more than 170 million people worldwide. Liver cancer and cirrhosis are two serious complications caused by HCV infection. Worldwide, the combination of alpha-interferon and ribavirin is a method of treating hepatitis C virus infection with an effective rate of approximately 40% [Jon Cohen. Science, 1999, 285: 26-30]. Hepatitis C virus is an enveloped single-stranded positive-strand RNA virus belonging to the Flaviviridae virus. The other two viruses of the Flaviviridae family are the dengue virus (Wang WK, et al. J Virol. 2002 May; 76(9): 4662-5.) and the Japanese encephalitis virus (Yun SI, et al. J Virol. 2003). Jun; 77 (ll): 6450-65.). Dengue virus and Japanese encephalitis virus are also single-stranded RNA viruses. Since no cell and animal models of hepatitis C virus have been studied, the results obtained by experiments using dengue virus and Japanese encephalitis virus can represent the results obtained by cell experiments with hepatitis C virus to a considerable extent.

Foot-and-mouth disease (FMD) is a potent infectious disease caused by foot-and-mouth disease virus. Pigs, cows, and sheep can be infected with foot-and-mouth disease virus and develop foot-and-mouth disease. In light, blisters, ulcers, and plaques appear in the mouth, tongue, lips, hooves, and breasts. Foot-and-mouth disease virus (FMDV) is the causative agent of foot-and-mouth disease. It is a member of the genus Aphthovims of the family Picornaviridae. Seven serotypes, namely 0, A, C, have been discovered. Type (called European type), SAT1, SAT2, SAT3 (South African 1, 2, 3, also known as African) and Asianl (Asian I, Asian). The occurrence and prevalence of foot-and-mouth disease can cause huge direct and indirect economic losses [Jiang Pengfei, Chinese Agricultural Sciences 1999, 32(6): 93~100]. The International Organization for Animal Health (OIE) lists foot and mouth disease as a Class A livestock infectious disease. All countries in the world attach great importance to the prevention and control of foot and mouth disease. In addition to the slaughter of livestock infected with foot-and-mouth disease, vaccination is the main measure to prevent and control foot-and-mouth disease [M. Woolhouse, 2001].

Sputum encephalitis is caused by the H encephalitis virus of the genus Flavivirus. Most people do not develop symptoms after infection with Japanese encephalitis virus. Most of the patients have mild clinical symptoms, such as fever, headache, nausea, burnout, abdominal pain or dysfunction and mental symptoms. In severe cases, sudden onset of high fever, headache, vomiting, followed by meningeal irritation, 3-5 days of paralysis, abnormal behavior, muscle rigidity, significant changes in consciousness, and even coma, death.

Hepatitis B virus is the causative agent of hepatitis B and is a DNA virus. The complete hepatitis B virus particle is 42 nm in diameter and can be divided into two parts: the envelope and the core. The protein on the envelope is called hepatitis B surface antigen (HBsAg), which is synthesized in hepatocytes and then released into the blood circulation, which is not contagious in itself. The core contains cyclic double-stranded DNA, DNA polymerase, core antigen (HBcAg) and e antigen (HBeAg), which are the mainstay of viral replication and are infectious. AIDS is a fatal infection caused by infection with human immunodeficiency virus (HIV). HIV mainly destroys the body's immune system, causing the body to gradually lose its defense ability and not be able to resist various pathogens from the outside world. HIV (HIV) is an enveloped RNA retrovirus that is classified as a retroviridae. HIV is spherical or ovoid, 100-130 nm in diameter, consisting of two parts, the envelope and the core. Envelope proteins include the outer membrane glycoprotein (gpl20) and the transmembrane glycoprotein (gp41). The core is composed of nucleocapsid protein, two identical copies of nucleic acid genomic RNA, and enzymes.

At present, the world has developed some drugs that can effectively suppress HIV. These drugs have been able to alleviate the symptoms of AIDS patients to some extent, prolong the lives of patients and improve their quality of life.

The papillomavirus belongs to the genus Papovaviridae of the papavirus family and includes papavirus and human papillomavirus (HPV) of various animals. HPV is a small DNA virus with a diameter of 45~55nm. The capsid is icosahedral in stereo symmetry, containing 72 shell particles and no capsule. The HPV genome is a closed-loop, double-stranded DNA with a molecular weight of 5 x 106 Daltons. According to the function, it can be divided into three areas: early zone (E zone), late zone (L zone) and non-coding zone (NCR). E is divided into E1~E7 幵 reading frames, which mainly encode proteins involved in viral replication, transcription, regulation and cell transformation. L distinguishes between L1 and L2, encoding the major capsid protein and the minor capsid protein, respectively. NCR is a DNA fragment of -6.4 to 1.0 bp in the E region and the L region, and is responsible for the regulation of transcription and replication. According to epidemiological survey data, the global infection rate of papillomavirus is about 9-13%, that is, about 630 million people are infected with papillomavirus every year. Papillomavirus is closely related to many skin and mucous membrane diseases. Infection of papillomavirus can lead to flat warts of the skin, genital warts, and more importantly, papillomavirus infection is closely related to cervical cancer, penile cancer and anal cancer (L Koutsky. Am J Med, May 5, 1997; 102(5 A): 3-8). Other studies have confirmed that herpes simplex virus type II and human papillomavirus are important biological factors leading to cervical cancer and the most common pathogens of reproductive system infection. The incidence of cervical cancer in women infected with cervical simple sore type 2 virus is 8 times higher than that in healthy women. Human papillomavirus is more likely to cause cervical cancer than herpes simplex type II virus. More than 100 types of human papillomavirus (HPV) have been identified (Brown DR. et al. J Clin icrohiol, 1999, 37(10): 3316-3322). Summary of the invention

One of the objects of the present invention is to provide a composition comprising a synthetic single-stranded deoxynucleotide and a ribavirin which is composed of one or more CpG-containing oligos The nucleotide single-stranded DNA molecule has the following structural formula (i)-(v;).

The artificially synthesized CpG-containing single-stranded deoxynucleotide of the present invention has the following structural formula: (1) (G)n(L)n X ₁ X ₂ CGYiY ₂ (M)n (G)n

3⁄4=A,T,G; X ₂ = A,T; Υ^ Α,Τ; Y ₂ = A,T,C; L,M=A,T,C,G; n is 0-6

5'-ggggTCgTTCgTCgTTgggggg-3' (SEQ ID NO: l)[121]

5'-ggg _g ATAACgTTgCggg _g gg-3' (SEQ ID NO: 2) [143]

5'-ggggTgCAACgTTCAgggggg-3' (SEQ ID NO: 3) [402]

5'-ggggTCCTACgTAggAgggggg-3' (SEQ ID NO: 4) [123]

5'-ggggTCCATgACgTTCCTgAAgggggg-3'(SEQ ID N0:5)[ 603] 5'-gggggACgTCgCCggggggg-3' (SEQ ID NO: 6) [118]

5'-ggATCCgTACgCATgggggg-3' (SEQ ID NO: 7) [320 ]

5'-gg _g ggAATCgATTCgggggg-3' (SEQ ID NO: 8) [154]

5'-gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 9) [464 ]

5'-ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 10) [471]

5'-gggACgTACgTCgggggg-3' (SEQ ID NO: 11) [390]

5'-gggggATCgACgTCgATC _ggg ggg-3' (SEQ ID NO: 12) [322]

5'-ggCgATCgATCgATCggggggg-3' (SEQ ID NO: 13) [333]

5'-ggggTCgATCgATCgAgggggg-3' (SEQ ID NO: 14) [113 ]

5'-ggTCgCgATCgCgAgggggg-3' (SEQ ID NO: 15) [307]

5 ' -ggGGTCAACGTTGAgggggG-3 ' (SEQ ID N0:16)[ 156 ]

5'-gTCgTTTTCgTCgACgAATTgggggggg-3' (SEQ ID NO: 17) [ 222 ] 5 '-gTCgTTATCgTTTTTTCgTAgggggg-3 ' (SEQ ID NO: 18) [151] 5'-ggCgTTAACgACgggggg-3' (SEQ ID NO: 19) [288 ]

5'-gTCggCACgCgACgggggg-3' (SEQ ID NO: 20) [157]

5'-ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 21) [ 312]

5 ' -gTCTATTTTgTACgTACgTgggg-3 ' (SEQ ID NO:22)[360 ]

5'-gACgTCgACgTCgACgTCAggggg-3' (SEQ ID NO: 23) [209]

5'-ggggTCgATCgTTgCTAgCgggggg-3' (SEQ ID NO:24)[399 ]

5'-gggggACgTTATCgTATTggggggg-3' (SEQ ID NO: 25) [600]

5'-ggggTCgTCgTTTgTCgTgTgTCgTTgggggg-3' (SEQ ID NO:26)[408] 5 '-ACgATCgATCgATCgggggg-3 ' (SEQ ID NO:27)[304]

5 '-AgACgTCTAACgTCggggg-3 ' (SEQ ID NO: 28) [301] 5'-ggggTgCTggCCgTCgTTgggggg-3' (SEQ ID NO: 29) [266] 5'-ggggTCgTTgCCgTCgggggg-3' (SEQ ID NO: 30) [248]

5'-ACCggTATCgATgCCggTgggggg-3' (SEQ ID NO: 31) [389 ] 5'-TTCgTTgCATCgATgCATCgTTgggggg-3' (SEQ ID NO: 32) [287]

(2) (G)n(L)nCG(XY)nCG(M)n(G)n

X=A,T; Y= A,T; L,M=A,T,C,G; n is 0-6

5'-ggggACgATACgTCggggggg-3' (SEQ ID NO: 33) [546 ]

5'-ggggACgATATCgATgggggg-3' (SEQ ID NO: 34) [1007]

5'-ggACgATCgATCgTgggggg-3' (SEQ ID NO: 35) [521]

5'-TCggggACgATCgTCgggggg-3' (SEQ ID NO: 36) [667]

5'- _ggg ggATCgATATCgATCgggggg-3' (SEQ ID NO: 37) [576] 5' - ggATC _g ATCgATCgAT _gg gggg-3' (SEQ ID NO: 38) [268]

5'-ggTgCATCgATCgATgCAgggggg-3' (SEQ ID NO: 39) [101] 5'-ggTgCATCgTACgATgCAgggggg-3' (SEQ ID NO: 40) [100] 5'-ggTgCgATCgATCgCAgggggg-3' (SEQ ID NO: 41) [134 ]

5'-gggggggTCgATCgATgggggg-3' (SEQ ID NO: 42) [ 519 ]

5'-ggggTCgTCgAACgTTgggggg-3' (SEQ ID NO: 43) [350]

5 ' -TgTCgTTCCTTgTCgTT-3 ' (SEQ ID NO: 44) [387]

5 '-TTCgCTTCgCTTTTCgCTTCgCTT-3 ' (SEQ ID NO: 45) [212] 5'-ACCgCCAAggAgAAgCCgCAggAggg-3' (SEQ ID NO: 46) [166] 5 '-TACAACggCgAggAATACC-3 ' (SEQ ID NO: 47) [ 176 ]

5 '-gTACAACggCgAggAATACCT-3 ' (SEQ ID NO: 48) [523] 5'-ACCgTCgTGgCCgTCggCCC-3' (SEQ ID NO: 49) [230]

5'-TgCTggCCgTCgTT-3' (SEQ ID NO: 50) [ 435 ]

5'-gTCggCACgCgACg-3' (SEQ ID NO: 51) [325 ]

5'-gTCggCACgCgACgCCCCCC-3' (SEQ ID NO: 52) [523]

5'-TCCCgCTggACgTT-3' (SEQ ID NO: 53) [188]

5 '-TTACCggTTAACgTTggCCggCC-3 ' (SEQ ID NO: 54) [403] 5 ' -ACCggTTAACgTTgTCCCCgggg-3 ' (SEQ ID NO: 55) [420] 5 '-CgTTgACgATCgTCCC ATggCggg-3 ' (SEQ ID NO: 56) [104] 5 '-TCTgCggCCTTCgTCg-3 ' (SEQ ID NO: 57) [ 257]

5'-TAgTAACCggTCCggCgCCCCC-3' (SEQ ID NO: 58) [221] 5'-TTgCAgCgCTgCCggTggg-3' (SEQ ID NO: 59) [611]

5'-CggCCCATCgAgggCgACggC-3' (SEQ ID NO: 60) [378]

5 '-TC ATCgACTCTCgAgCgTTC-3 ' (SEQ ID NO: 61) [599]

5 ' - ATCgTCgACTCTCgTgTTCTC-3 ' (SEQ ID NO: 62) [201] 5 '-TgCAgCTTgCTgCTTgCTgCTTC-3 ' (SEQ ID NO: 63) [153] 5 '-ggTgCgACgTCgCAgATgAT-3 ' (SEQ ID NO: 64) [116 ]

5 ' -ggTCgAACgTTCgAgATgAT-3 ' (SEQ ID NO:65)[133]

5 '-gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 66) [278] 5 ' -actcgagacgcccgttgatagctt-3 ' (SEQ ID NO: 67) 355 [244]

5'-AACgTTggCgTCgACgTCAgCgCC-3' (SEQ ID NO: 68) [623] 5 '-gACgTCgACgTTgACgCT-3 ' (SEQ ID NO: 69) [485]

5 '-ggCgTTAACgTTAgCgCT-3' (SEQ ID NO: 70) [579]

5 '-AgCgCTAgCgCTgACgTT-3 ' (SEQ ID NO:71)[232]

5 '-CTAgACgTTC AAgCgTT-3 ' (SEQ ID NO: 72) [233]

5'-gACgATCgTCgACgATCgTC-3 ' (SEQ ID NO: 73) [344]

5 ⁵ -gTCgTTCgTAgTCgACTACgAgTT-3 ' (SEQ ID NO: 74) [379] 5 ' - AAAAgACgTCgACgTCgACgTCTTTT-3 ' (SEQ ID NO: 75) [489] 5 ' -TgCgACgATCgTCgCACgATCggAT-3 ' (SEQ ID NO: 76) [479 ] 5 '-TgCgACgTCgCAC AgCgT-3 ' (SEQ ID NO: 77) [492]

(3) (TCG)n(L)nCG (M)n(G)n

L, M = A, T, C, G; n is 0-6

5'-TCgTTgCCgTCgg-3' (SEQ ID O: 78) [619]

5'-TCgTTgCCgTCggg-3' (SEQ ID NO: 79) [577]

5'-TCgTTgCCgTCgggg-3' (SEQ ID NO: 80) [533]

5'-TCgTTgCCgTCggggg-3' (SEQ ID NO: 81) [537]

5'-TCgTTgCCgTCgggggg-3' (SEQ ID NO: 82) [481] 5,-TCgTTgCC _g TCggggggg-3, (SEQ ID NO: 83) [ 177 ] 5'-TCgTTgCCgTCgggggggg-3' (SEQ ID NO: 84) [lll]

5'-TCgTTgCCgTCggggggggg-3' (SEQ ID NO: 85) [105]

5'-TCgTCgggTgCATCgATgCAgggggg-3' (SEQ ID NO: 86) [664] 5'-TCgTCgggTgCAACgTTgCAgggggg-3' (SEQ ID NO: 87) [564] 5'-TCgTCgggTgCgTCgACgCAgggggg-3' (SEQ ID NO: 88) [542 ] 5'-TCgTCgggTgCgATCgCAgggggg-3' (SEQ ID NO: 89) [450]

5'-TCgTCgggTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO: 90) [465] 5'-TCgTCgTgCgACgTCgCAgggggg-3' (SEQ ID NO: 91) [498]

5'-TCgTCgCAgAACgTTCTgggggg-3' (SEQ ID NO: 92) [527]

5'-TCgTgCgACgTCgCAgggggg-3' (SEQ ID NO: 93) [112]

5'-TCgTgCgACgATCgTCgCAgggggg-3' (SEQ ID NO: 94) [178] 5'-TCgTATgCATCgATgCATAgggAgg-3' (SEQ ID NO: 95) [410] 5 '-TCgTgCATCgATgCAgggggg-3 ' (SEQ ID NO: 96) [ 444 ]

5'-TCgAAACgTTTCgggggg-3' (SEQ ID NO:97)[532]

5'-TCggACgATCgTCgggggg-3' (SEQ ID NO: 98) [598]

5'-TCgAgCgATCgCTCgAgggggg-3' (SEQ ID NO: 99) [555]

5 '-TCgTCgCTTTgTCgTTgggg-3 ' (SEQ ID NO: 100) [418]

5'-TCgTCgTTTTgTCgTTgggg-3, (SEQ ID NO: 101) [208]

5'-TCgTCgggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 102) [302] 5'-TCgTCgggTgCgACgATCgTCgggggg-3' (SEQ ID NO: 103) [290] 5'-TCgTCgTTTgCATCgATgCAggggggg-3' (SEQ ID NO: 104) [627 ] 5 ' -TCgTCgTTTTgACgATCgTCgggggg-3 ' (SEQ ID NO: 105) [500] 5'-TCgTTCggggTgCCg-3' (SEQ ID NO: 106) [103]

5 ' -TCgTTCggggTACCgATgggg-3 ' (SEQ ID NO: 107) [578]

5'-TCgTTgCgCTCCCATgCCgggggg-3' (SEQ ID NO: 108) [319] 5'-TCgTCgTTTCgTCgTTgggg-3' (SEQ ID NO: 109) [647]

5 '-TCgTTgTCgTTTCgCTgCCggCggggg-3 ' (SEQ ID NO: 110) [417] 5'-TgCTTgggTggCAgCTgCCAgggggg-3' (SEQ ID NO: lll) [427] 5 ' -TgCTgCTTTgCTgCTTgggg-3 ' (SEQ ID NO: 112) [421 ] 5'-AACgTTCgACgTCgAACggggggg-3' (SEQ ID NO: 113) [453]

5'-AACgACgACgTTggggg-3' (SEQ ID NO: 114) [580]

(4) (TCG)n(L)nX ₁ X ₂ CG (M)n

X^=A,T,G; X ₂ =A,T; L,M=A,T,C,G; n is 0-6

· The design sequence is as follows:

5 '-TCgTAACgTTgTTTTTAACgTT-3 ' (SEQ ID NO: 115) [470]

5 '-TCgTCgTATACgACgATCgTT-3 ' (SEQ ID NO: 116) [502]

5'-TCgTCgTTTgCgTTgTCgTT-3' (SEQ ID NO: 117) [601]

5 ' -TCCTgTCgTTTTgTCgTT-3 ' (SEQ ID NO:118)[625]

5 ' -TCgTCgTTgTCgTTCgCT-3 ' (SEQ ID NO:119)[430]

5 '-TCgTCgTTACCgATgACgTCgCCgT-3 ' (SEQ ID NO: 120) [480] 5 ' -TCgTCgTTTgC ATCgATgC AgTCgTCgTT-3 ' (SEQ ID NO: 121) [108] 5 ' -TCgCCTCgTCgCCTTCgAgCg-3 ' (SEQ ID NO: 122) [102]

5'-TCgTgTgCgTgCCgTTgggT-3' (SEQ ID NO: 123) [406]

5'-TCgTCgAgggCgCCggTgAC-3' (SEQ ID NO: 124) [560]

5,-TCgTCgCCggTgggg _g TgTg-3, (SEQ ID NO: 125) [629]

5 '-TCgTCgTACgCAATTgTCTT-3 ' (SEQ ID NO: 126) [440]

5'-TCgCCCACCggTgggggggg-3' (SEQ ID NO: 127) [207]

5'-TCgTCgCAgACCggTCTgggg-3' (SEQ ID NO: 128) [615]

5 '-TCgTCgCggCCggCgCCCCC-3 ' (SEQ ID NO: 129) [610]

5'-TCgTCgCggCCgCgAggggg-3' (SEQ ID NO: 130) [206]

5 ' -TCgAggAC AAgATTCTCgTgC-3 ' (SEQ ID NO: 131)[ 119]

5 '-TCgAggAC AAgATTCTCgTgCAggCC-3' (SEQ ID NO: 132) [570] 5 '-TCgTgC AggCCAACgAggCCg-3 ' (SEQ ID NO: 133) [631]

5'-TCgTTgCCgTCggCCC-3' (SEQ ID NO: 134) [115]

5 '-TCggCACgCgACgTgCTggCCgTCgTTTCC-3 ' (SEQ ID NO: 135) [370] 5'-TCgTTgCCgTCggCCCCCCCCC-3' (SEQ ID NO: 136) [309]

5'-TCgTTgCCgTCggCCCCCC-3' (SEQ ID NO: 137) [506]

5 '-TCgTTgCCgTCggCCCCC-3 ' (SEQ ID NO: 138) [ 404 ]

5 '-TCgTTgCCgTCggCCCC-3 ' (SEQ ID NO: 139) [203] )][(Q6S ID NO85E

)]] gQgggg (g ID NO6C3SE84cACTCTCTTCTTTC 5TT- ,

〕) [gggQ (7〇:27E ID NC1rCTCACCTTCTTC3S 5TTT--">

QSE IDNOCGGGGACC (TCGTGCGTGCATT- 】gggggg (QOC TE ID N:TCTTriTrTriST- ..

:]gggg (Q50 NO:5CTCTnrCCrTCC3S ID 255T1TTET---,,,

)[]gggg (Q7 NO40CACTTCTCTTSE ID 5TTCTTT

〕 gXggg (Q 164CT3 ID NO: 250TCTTTTCTCTCSE 5TT- [] ggg) (Q65S I NO0cTCTTCACTCTTTC3ED 5TCT- ,

〕ggggg (Q 160CCC3 I NO:330TACTCTATTSED 5 T-"

)[gggg (QCCD NOTCTTTTCAAATTSE IT- 〕)) [0 NO31

)[jgg (gggggggggQD NCCCCrSE IO656TCTACTTT 5T-- ) [] (QE ID NO380S

)[ggggg (Q]gggggD NO607CTCCCCTACCSE IACT- )[]ggggg (gQcC NOTTCTACATSE IDT

)[]gggggg (QO5cCS N40TTCTAATT3E ID 5T- ]) [ggggg (gQ528C I NO:150cTTCTAAATTSED 5T-,>

〕g)[ggggg (Q940CCE IOTTCTCSD N T- ) []ggggggg (QgggD0SE I NO:15640CCCTTCT3TCTTTT 5T--"

)_:]gggggg (QOD N306CCTTTCTS ITCTCTTET- )[]ggggg (QSOcTCTTATTT3E ID N 5TT- - X ()gggggQggggggO:70SE ID N 1425CTCAACTCCATTTT 5T- []ggg ()gggggggQ8 ID NO20TECTCACTCTCS yT- )[]gg (gggggggQ NOcCTTCSEDTCc IT ->

)[]ggggggg (ggQ 106T ID NOCTCTCCT3SETT 5-"

]))[ggggggg (QgggO630 ID NCTCCTCAC: TSE 5CCT- [] (Q)ggggggggggSEO:59TCT3 ID N1420CCACCTCTcT 5CACT---"

〕) [gQgggg (TTTD NO 305CACAAACCTTSE IA T-X]ggggg (Q ID §: 15CTCCCCCCE4001CTTCCC3S 5T- - 5'-TCgTgCgACgTCgCAgATgAT-3' (SEQ ID NO: 169) [114]

5 '-TCgTCgAgCgCTCgATCggAT-3 ' (SEQ ID NO:170)[211 ]

5 '-TCgTCgTTTCgTAgTCgTTgACgTCggg-3 ' (SEQ ID NO: 171) [204]

5 '-TCgTCggACgTTTTCCgACgTTCT-3 ' (SEQ ID NO: 172) [308]

5 '-TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 173) [340]

5 ' -TCgTCgTTTgTCgTgTgTCgTT-3 ' (SEQ ID NO:174)[503]

5'-TCgTCgTTggTCggggTCgTTggggTCgTT-3' (SEQ ID NO: 175) [405]

5'-TCgTCgTTTCgTCTCTCgTT-3' (SEQ ID NO: 176) [614]

5 '-TCgTCgTTTTgCTgCgTCgTT-3 ' (SEQ ID NO: 177) [ 505]

5 ' -TCgAgCgTTTTCgCTCgAATT-3 ' (SEQ ID NO:178)[530 ]

(5) Sequence containing TTCGTCG

5'-TTCgTCgTTTgATCgATgTTCgTTgggggg-3' (SEQ ID NO:179)[ 507 ]

5'-TTCgTCgTTgTgATCgATgggggg-3'-3' (SEQ ID NO:180)[ 210 ]

5'-TATCgATgTTTTCgTCgTCgTTgggggg-3' (SEQ ID NO: 181) [202]

5 '-TCgACTTTCgTCgTTCTgTT-3 ' (SEQ ID NO: 182) [303]

5'-TCgTCgTTTCgTCgTTCTC-3' (SEQ ID NO: 183) [ 491]

5'-TCgACgTTCgTCgTTCgTCgTTC-3 ' (SEQ ID NO: 184) [590]

5 ' -TCgTCgTTTTCgTCgTTTTCgTCgTT-3 ' (SEQ ID NO: 185) [633]

Preferably, the synthetic CpG-containing single-stranded deoxynucleotide of the present invention has the sequence shown below

CpG ODN 1: 5'-ggggacgatatcgatgggggg-3' SEQ ID NO 34

CpG ODN 2: 5 ' -ggggtcgttcgtcgttgggggg-3 ' SEQ ID NO 1

CpG ODN 3: 5 ' -ggggtgcaacgttcagggggg-3 ' SEQ ID NO 3

CpG ODN 4: 5, -tcgtgcgacgatcgtcgcagggggg-3 ' SEQ ID NO 94

CpG ODN 5: 5 ' -tcgagcgatcgctcgagggggg-3 ' SEQ ID NO 99

CpG ODN 6: 5 ' -ggggtgctggccgtcgttgggggg-3 ' SEQ ID NO 29

CpG ODN 7: 5 ' -tcgaaacgtttcgggggg-3 ' SEQ ID NO 97

CpG ODN 8: 5 '-tcgtcgggtgcgacgtcgcagggggg-3 ' SEQ ID NO 102

CpG ODN 9: 5 ' -tcggacgatcgtcgggggg-3 ' SEQ ID NO 98

CpG ODN 10 5 ' -tcgtcgggtgcgatcgcagggggg-3 ' SEQ ID NO 89

CpG ODN 11 5 ' -tcgtgcgacgtcgcagatgat-3 ' SEQ ID NO 169 CpG ODN 12: 5 ' -tcgtatgcatcgatgcatagggagg-3 ' SEQ ID NO 95

CpG ODN 13: 5 ' -tcgtcgtttcgtcgttgggg-3 ' SEQ ID NO 109

CpG ODN 14: 5 '-tcgtcgggtgcatcgatgcagggggg-3 ' SEQ ID NO 86

CpG ODN 15: 5 ' -tcgtcgcagaacgttctgggggg-3 ' SEQ ID NO 92

CpG ODN 16: 5 ' -tcgtcgggtgcgacgtcgca-3 ' SEQ ID NO 186

CpG ODN 17: 5'-gtcgttttcgtcgacgaattgggggggg-3' SEQ ID NO 17

CpG ODN 18: 5 ' -tcgtcgtcgactcgtggtcggggg-3 ' SEQ ID NO 157

CpG ODN 19: 5 '-tcggggacgatcgtcgggggg-3 ' SEQ ID NO 36

CpG ODN 20: 5 ' -gggggcgtcgttttcgtcgacgaatt-3 ' SEQ ID NO 66 One of ordinary skill in the art, based on the teachings of the specification, in conjunction with common general knowledge in the art, is well established that the above-described synthetic single-stranded deoxynucleotides can be used. The object of the invention is achieved.

The artificially synthesized single-stranded deoxynucleotides of the present invention can be produced by a known method, for example, by a solid phase phosphoramidite triester method. A method for producing the artificially synthesized CpG-containing single-stranded deoxyoligonucleotide of the present invention is exemplified in detail in the following examples.

One of the objects of the present invention is to provide a composition for treating or preventing a viral infection and a related disease caused by a viral infection, the composition comprising ribavirin and a synthetic CpG-containing single-stranded deoxynucleotide. The artificially synthesized single-stranded deoxynucleotide is composed of an oligonucleotide single-stranded DNA molecule containing one or more CpGs, and has a sequence contained in one of the formulas (i) to (v). The phosphodiester bond of a synthetic CpG-containing single-stranded deoxynucleotide is non-vulcanized, partially vulcanized or fully vulcanized, and can be chemically modified.

A second object of the present invention is to provide a method for the combined use of artificially synthesized CpG-containing single-stranded deoxynucleotides and ribavirin for treating or preventing diseases caused by viral infections and viral infections, and artificial synthetic The phosphodiester bond of the single-stranded deoxynucleotide of CpG is non-vulcanized, partially vulcanized or fully vulcanized and can be chemically modified.

A third object of the present invention is to provide a method of treating a viral infection and a viral infection-associated disease comprising administering to a patient in need of such treatment a therapeutically effective amount of a human having a sequence of structural formula (i)-(v) Synthetic single-stranded deoxynucleotides and ribavirin. Both can be administered simultaneously, sequentially or in separate doses. The virus refers to RA virus, DNA virus and retrovirus, including but not limited to influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus and nipple Tumor virus. · These synthetic single-stranded deoxynucleotides are administered at a single dose of 1-5000 micrograms in the prevention and treatment of diseases caused by viral infections and viral infections. A single application dose of ribavirin is 300-10000 mg.

The application of synthetic single-stranded deoxynucleotides and ribavirin includes mucosal surfaces (including respiratory, digestive tract and genitourinary mucosa) applications, subcutaneous, intramuscular injection, gastrointestinal applications, abdominal applications, intravenous injections, etc. Way to apply.

A fourth object of the present invention is to provide a method of treating or preventing a viral infection and a related disease caused by a viral infection comprising administering to a patient in need of such treatment a therapeutically effective amount of the above composition.

A fifth object of the present invention is to provide use of the above composition for the preparation of a medicament for treating or preventing a viral infection and a disease associated with viral infection.

The phosphodiester bond of the artificially synthesized single-stranded deoxynucleotide in the present invention may be non-vulcanized, partially vulcanized, or fully vulcanized. Synthetic single-stranded deoxynucleotides can be chemically modified. The chemical modifications described therein are well known to those of ordinary skill in the art.

In the present invention, the virus includes, but is not limited to, influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and papillomavirus. In addition, it should be noted that other aspects of the present invention and the inventive benefits of the present invention can be directly inferred by those skilled in the art based on the disclosure of the present application. detailed description

In the context of the present invention, the terms used generally have the meanings as commonly understood by one of ordinary skill in the art, unless otherwise indicated. The invention is described in further detail below with reference to the specific examples of the preparations and the biological effects of the invention. It is to be understood that the invention is not intended to limit the scope of the invention in any way. Example

In the following examples, various processes and methods not described in detail are conventional methods well known in the art, such as the synthesis using a solid phase phosphoramidite triester method.

In the following examples, the source of the reagent used, the trade name, and/or the necessity to list its constituents, They are only marked once. The same reagents used hereinafter are not described above unless otherwise stated. Example 1. Preparation of Synthetic Single-Strand Deoxynucleotides

Synthesis of artificially synthesized single-stranded deoxynucleotides by solid phase phosphoramidite triester

1) Deprotection group

The protecting group dimethoxytrityl (DMT) of the nucleotide attached to the controlled glass (Controlled Pore Glass) was removed by Trichloroacetic Acid (TC A) to obtain a free 5'. - a hydroxyl end for the next condensation reaction.

2) Activation

The phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protected), this intermediate will undergo a condensation reaction with the deprotected nucleotide on the controlled porous glass.

3) Connection

When a phosphoramidite tetrazole reactive intermediate encounters a deprotected nucleotide on a controlled porous glass,

The 5'-hydroxy group undergoes an affinity reaction, condenses and removes the tetrazole, and the synthesized oligonucleotide chain is extended by one base forward.

4) Closed

After the condensation reaction, in order to prevent the 5'-hydroxyl group which is not involved in the reaction on the controlled porous glass from being extended in the subsequent cyclic reaction, the terminal hydroxyl group is often blocked by acetylation, and the general acetylating reagent is acetic anhydride and N. - Formed by the mixing of methyl imidazole.

5) oxidation

In the condensation reaction, the nucleoside monomer is linked to the oligonucleotide attached to the controlled porous glass through the phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by acid or alkali. At this time, iodine tetrahydrofuran is commonly used. The solution converts the phosphorous amide to a phosphate triester to give a stable oligonucleotide.

After the above five steps, a deoxynucleotide is attached to the nucleotide of the controlled porous glass, and the protective group on the newly added deoxynucleotide 5'-hydroxyl group is also removed by trichloroacetic acid. After the DMT is repeated, the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment. Finally, it is cleaved and deprotected (generally, C base is protected with benzoyl; G base is protected with isobutyryl; T base is not protected; phosphorous acid is protected with nitrile ethyl), purified (commonly used The HAP, PAGE, HPLC, C18, OPC and other methods, quantitative and other synthetic post-treatment can obtain oligonucleotide fragments that meet the experimental requirements. Unvulcanized synthetic single-stranded deoxynucleotides were synthesized on an ABI 3900 DNA synthesizer; the synthesis of fully sulfurized and partially sulfurized CpG single-stranded deoxyoligonucleotides was synthesized on an ABI 394 DNA synthesizer using a displacement method.

Example 2, Example 3, Example 4, Example 5 The artificially synthesized single-chain deoxynucleotide used in Example 6 was CpG ODN 1-20 described above. EXAMPLES 2. Comparison of anti-follicular inflammatory virus effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides in combination with ribavirin

1. Expansion of follicular inflammatory virus (VSV)

1) Equipment and materials: low temperature refrigerator, carbon dioxide incubator, ultra clean bench, inverted microscope, liquid nitrogen tank, distilled water, vacuum pump, cell culture flask, pipette of various specifications, pipette, dropper, etc.

2) IMDM medium: IMDM (GIBCOBRL) containing L-glutamine 10.4 g, 2.0 g of sodium bicarbonate, 100,000 units of gentamicin, plus three distilled water to a volume of 1000 ml. The 0.22 micron filter vacuum pump was used for filtration and sterilization.

3) Method: Vero E6 cells (purchased from Shanghai Cell Institute) were cultured in IMDM medium containing 10% calf serum (Invitrogen, treated at 56 C for 30 minutes). After growing into a single layer in a 100 ml culture flask, the culture solution was changed. A culture solution containing a follicular inflammatory virus (VSV) (Changchun Bioproducts Institute) was added at a level of 10 TCID50. Set no virus control. After about 24-48 hours, all of the Vero E6 cells infected with VSV developed lesions, and the diseased cells and supernatant were collected and allowed to stand at 20 ° C overnight. The next morning, melt it, slam it with a straw, and centrifuge to collect the supernatant. After the TCID50 was measured, the supernatant was stored at 20 ° C for use.

2, determination of follicular inflammatory virus (VSV) TCID50

1) Equipment and materials: Same as above.

2) IMDM medium: Same as above.

3) IMDM medium containing 10% calf serum: 10 ml of calf serum (Invitrogen, treated at 56 ° C for 30 minutes), 90 ml of IMDM medium.

4) 0.5% crystal violet dye solution: Crystal violet 0.5 g, NaCl 0.85 g, dissolved in 50 ml of anhydrous ether. Add 3 ml of formaldehyde and 47 ml of distilled water.

5) Crystal violet decolorizing solution: 50 ml of ethylene glycol monomethyl ether is mixed with 50 ml of distilled water.

6) 0.25 % trypsin (Trypsin)

7) Method: Digest well-grown Vei-o E6 cells with 0.25 % trypsin for 3-5 minutes. Centrifugation The cells were washed (1000 rpm, 5 minutes). The cell concentration was adjusted to ⁴ x 10 ⁵ /ml with IMDM medium of 10% calf serum. The cell suspension was added to a 96-well flat-bottomed plate (Costar) at 100 μL per well with three replicate wells. After 24 hours of incubation at 37 ° C in a C0 ₂ incubator, the cells formed a monolayer. Replace the culture solution, which contains the diluted VSV (Changchun Bioproducts Institute). Set no VSV control. After 24 hours, the degree of cytopathic effect was determined by staining with crystal violet. The culture solution was aspirated, and 200 μl of 0.5% crystal violet dye solution was added to each well at 37 ° C for 15 minutes. The running water washes away the crystal violet dye solution. Add 200 μl of crystal violet decolorizing solution to each well, shake the oscillator to completely remove the dye from the cells, and use a spectrophotometer at 540 nm wavelength (Cytokines. A practical approach, second edition, edited by RR. BAL WILL. The practical approach series.). The reciprocal X 10 of the virus dilution with 50% cytopathic effect was the TCID50 I ml value for this viral solution.

3. Separation of human peripheral blood mononuclear cells

1) Equipment and equipment: low temperature refrigerator, carbon dioxide incubator, ultra clean bench, inverted microscope, liquid nitrogen tank, distilled water, vacuum pump, cell culture flask, bacteria filter, filter bottle, various specifications of straw, plus Samples, droppers, blood cell counting plates, horizontal centrifuges, etc.

2) Reagents and materials: Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun. Polysucrose - diatrizoate lymphocyte stratification: specific gravity 1.077 ± 0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. IMDM medium: Same as above.

3) Method: Separation of human peripheral blood mononuclear cells with polysucrose-diazepam lymphocyte stratification solution (Beijing Dingguo Biotechnology Co., Ltd.). The volume ratio of stratified fluid to heparin anticoagulated peripheral blood is approximately 2:1. Centrifuge horizontally (1,000 X g, 20 min). Pipette the liquid containing the mononuclear cells with a pipette and place them in another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at 1,000 x g for 15 min and discard the supernatant. Repeat the wash twice. The supernatant was discarded, the cells were resuspended in 2 ml of culture medium, and cell counts were performed.

4. Determination of the effect of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin on anti-follicular inflammatory virus

The final concentration of human peripheral blood mononuclear cells (PBMC) was adjusted to ³ x 106/ml using IMDM medium containing 10% calf serum. Add cell suspension to a 12-well culture plate at 2 ml per well. Add CpG ODN (l-20) to a final concentration of 6 g/ml. The supernatant was collected by incubating at 37 ° C, 5 % carbon dioxide incubator for 48 hours.

CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established. Vero E6 cells in good growth were seeded in 96-well plates at 1.3 X 10 ⁴ cells per well, 37 ° C, 5% After 24 hours of C0 ₂ culture, the CpGODN group was supplemented with a 1:100 dilution of CpG ODN-stimulated human PBMC culture supernatant, and the ribavirin group was added with ribavirin (to a final concentration of 10 μmol/l), CpGODN + ribavi The forest group was supplemented with 1:100 diluted CpG ODN-stimulated human PBMC culture supernatant and ribavirin (to a final concentration of 10 μmol/1) (Zhejiang Chengyi Pharmaceutical Co., Ltd.), and the IMDM group only added the same amount of IMDM. After continuing to culture for 1.5 hours, 250 TCID50 I ml of VSV virus was added, and a normal VERO cell control group was set and cultured for 44 hours. The degree of cytopathic effect was determined by staining with crystal violet. Aspirate the culture solution. Add 200 μl of 0.5% crystal violet stain to each well and incubate for 15 minutes at 37 °C. The running water washes away the crystal violet dye solution. 200 microliters of crystal violet decolorizing solution was added to each well, and the shaker was shaken to completely remove the dye from the cells, and the spectrophotometric value was measured by a spectrophotometer at a wavelength of 540 nm.

5. Results: Compared with the combination of CpG ODN and CpG ODN and ribavirin, the OD value of CpG ODN and ribavirin combined application group was significantly higher than that of CpG ODN group. Single-stranded deoxynucleotides have significant antiviral effects, and the combination of synthetic single-stranded deoxynucleotides and ribavirin has a more significant antiviral effect. The comparison of OD values between the two groups is shown in Table 1. Example 3 Comparison of anti-influenza virus effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides N combined with ribavirin

1. Separation of human peripheral blood mononuclear cells

1) Apparatus and equipment: Same as part 3 (1) of Example 2.

2) Reagents and materials: Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun. Polysucrose - diatrizoate: Specific gravity 1.077 ± 0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. DMEM medium: 1000 ml containing 100,000 units of gentamicin. The 0.22 micron filter was sterilized by filtration and dispensed.

3) Method: Mononuclear cells of human peripheral blood were isolated using a sucrose-diatrizoate lymphocyte stratified solution. The volume ratio of the stratified fluid to the heparin anticoagulated peripheral blood is about 2:1. Centrifuge horizontally (1,000 X g, 20 min). Pipette the liquid containing the mononuclear cells with a pipette and place in a separate centrifuge tube. Add an equal volume of serum-free medium. 1,000 g Centrifugal for 15 min, discard the supernatant. Repeat the wash twice. Discard the supernatant, resuspend the cells in 2 ml of culture medium, and perform cell counting.

2. Determination of anti-influenza virus effect by synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin The final concentration of human peripheral blood mononuclear cells was adjusted to 3χ10 ^δ /ml using DMEM culture medium. This cell suspension was added to a 12-well culture plate at 2 ml per well. Synthetic single-stranded deoxynucleotides were added to a final concentration of 6 g/ml. The cells were cultured in a 5% carbon dioxide incubator at 37 ° C for 48 hours, and the supernatant was collected and tested for activity against influenza virus.

CpGODN group, ribavirin group, CpGODN+ribavirin group and DMEM control group were established. The concentration of VeroE6 cells in a well-growth state was adjusted to 3χ10 ⁵ /ml with 0.3% BSADMEM. The cells were seeded in 96-well flat-bottomed plates at 1.3×10 ⁴ cells per well, cultured at 37° C. and 5% CO _{2 for} 24 hours, and the CpG group was stimulated with a 1:100 dilution of synthetic single-stranded deoxynucleotides. Human PBMC culture supernatant, ribavirin group was added to ribavirin (to a final concentration of 10 μιηο1/1), and CpG+ribavirin group was supplemented with 1:100 dilution of synthetic single-stranded deoxynucleotide-stimulated human PBMC. The supernatant and ribavirin were cultured (to a final concentration of 10 μmol/1) (Zhejiang Chengyi Pharmaceutical Co., Ltd.), and only DMEM was added to the DMEM group. After continuing to culture for 1.5 hours, 0.3 μ of BSA DMEM (4 wg/ml trypsin) diluted influenza virus solution (Changchun Institute of Biological Products) was added to each well 200 μl, and a normal VERO cell control was set. The cells were cultured in a 37 ° C, 5% CO ₂ incubator for 44 hours, and the degree of cytopathic effect was determined by crystal violet staining. Aspirate the culture solution. Add 200 μl of 0.5% crystal violet dye solution to each well at 37 ° C for 15 minutes. The running water washes away the crystal violet dye solution. 200 microliters of crystal violet decolorizing solution was added to each well, and the shaker was shaken to completely remove the dye from the cells, and the spectrophotometric value was measured by a spectrophotometer at a wavelength of 540 nM.

3 Results: Compared with the combination of CpGODN and CpGODN and ribavirin, the OD value of CpG ODN and ribavirin combined application group was significantly higher than that of CpG ODN group. This result indicated that synthetic single-chain deoxygenation Nucleotides have a significant anti-influenza effect, and the combination of synthetic single-stranded deoxynucleotides and ribavirin has a more pronounced anti-influenza effect. The OD values between the two groups are shown in Table 2. Example 4. In vitro experiments with synthetic single-stranded deoxynucleotides and ribavirin against influenza virus in mice

1 Materials: Female Kunming mice, 14-16 grams, 6-8 weeks old, provided by the animal room of Jilin University. Influenza virus H1N1 lung-adapted strain (FM1) was purchased from Changchun Institute of Biological Products.

2 Methods: Normal mouse control, model control group (negative control group), ribavirin group, CpGODN group, ribavirin plus CpG ODN group were established. The CpGODN group was injected subcutaneously with artificially synthesized single-stranded deoxynucleotide 80Pg (CpG ODN 1-20). After 48 hours, the influenza virus was diluted to 0.3 LD ₅ Q with 0.3% BSA DMEM, except for the normal mouse control group. , other groups of mice were anesthetized with ether, nasally given 20μ1 of 10 LD _5Q flu virus. Two hours later, mice in the ribavirin group were administered by gavage, 50 mg ribavirin/kg body weight, 0.2-0.4 mI per mouse, 3 times daily. 80 wg CpG ODN was administered to mice in the CpGODN group by subcutaneous injection, once every two days. Ribavirin plus CpGODN group, ribavirin and CpGODN were administered at the same dose and administration as the ribavirin group and the CpGODN group. Four days later, the mice were dissected, the lungs were taken, weighed, and the lung lesion index was determined. Pulmonary Lesion Index (Biochemical and Biophysical Research Communications 279, 158-161 (2000). Inhibitory Effects of an Antisense Oligonucleotide in an Experimentally Infected Mouse Model of Influenza A Virus.)

No obvious lesions

+ 25 % lung tissue lesions

+ + 25-50% lung lesions

+ + + 50-75% lung lesions

+ + + + >75 % lung lesions 3. Results: The lung lesion index of the CpGODN+ ribavirin group was significantly lower than that of the CpGODN group and the ribavirin group. This indicates that the synthetic single-stranded deoxynucleotide combined with ribavirin has better effects than the synthetic single-stranded deoxynucleotides and ribavirin alone. The comparison of lung lesion index between groups is shown in Table 3. 4. Conclusion: Synthetic single-stranded deoxynucleotides have obvious anti-influenza virus effect, and the combined use of synthetic single-chain deoxynucleotides and ribavirin has a more significant anti-influenza virus effect. Example 5. Comparison of CpGODN-synthesized single-stranded deoxynucleotides and CpGODN-synthesized single-stranded deoxyribonucleic acid combined with ribavirin for anti-foot-and-mouth disease virus

1. Separation of human peripheral blood mononuclear cells

1) Equipment and equipment: Same as above.

2) Reagents and materials: Foot-and-mouth disease virus type 0 is provided by Inner Mongolia Biopharmaceutical Factory, and anti-virus experiment is carried out at Inner Mongolia Bio-Pharmaceutical Factory. Heparin anticoagulated human whole blood was purchased from the blood station in downtown Changchun. Polysucrose - diatrizoate: specific weight 1.077 ± 0.001, purchased from Beijing Dingguo Biotechnology Co., Ltd. IMDM medium: 1000 ml containing 100,000 units of gentamicin. The 0.22 micron filter was sterilized by filtration and dispensed.

3) Method: Mononuclear cells of human peripheral blood were isolated using a sucrose-diatrizoate lymphocyte stratified solution. The volume ratio of stratified fluid to heparin anticoagulated peripheral blood is about 2:1. Centrifuge horizontally (1,000 X g, 20 min). Using a straw Pipette the liquid containing the mononuclear cells and place them in another centrifuge tube. Add an equal volume of serum-free medium. Centrifuge at 1,000 x g for 15 min and discard the supernatant. Repeat the wash twice. The supernatant was discarded, and the cells were resuspended in 2 ml of medium and subjected to cell counting. 2. Determination of anti-foot-and-mouth disease virus combined with synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin

Mediator IMDM culture liquid containing 10% fetal bovine peripheral blood mononuclear cells at a final concentration 3x l0 ⁶ cells / ml. Add this cell suspension to a 12-well culture plate at 2 ml per well. Add synthetic single-stranded deoxynucleotides to a final concentration of 6 μ _§ /πι1. The 3TC, 5 % carbon dioxide incubator was cultured for 48 hours, and the supernatant was collected and tested for activity against foot-and-mouth disease virus.

CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established. The concentration of BHK cells with good growth was adjusted to ³ x 105/ml with 5% fetal bovine serum IMDM. The cells were seeded in a 96-well flat-bottomed plate, 1.3×10 ⁴ cells per well, cultured at 37 ° C, 5% CO _{2 for} 24 hours, and the CpGODN group was spiked with a 1:100 dilution of synthetic single-stranded deoxynucleotide. Human PBMC culture supernatant, ribavirin group added to ribavirin (to a final concentration of μ πιοΙ / l), CpG ODN + ribavirin group added 1: 100 dilution of CpG OD stimulated human PBMC culture supernatant and benefit Baverin (Zhejiang Chengyi Pharmaceutical Co., Ltd.), IMDM group only added the same amount of IMDM. After 1.5 hours of incubation, 2% fetal bovine serum was added to each well, and IMDM diluted foot-and-mouth disease virus solution (Inner Mongolia Biopharmaceutical Factory) 200 μl, and normal Vera cell control was set. 37 ^Q C, 5 % C0 ₂ incubate for 44 hours, and the degree of cytopathic effect was determined by crystal violet staining. Aspirate the culture solution. Add 200 μl of 0.5% crystal violet stain to each well at 37 ° C for 15 minutes. The running water washes away the crystal violet dye solution. 200 microliters of crystal violet decolorizing solution was added to each well, and the shaker was shaken to completely remove the dye from the cells, and the spectrophotometric value was measured by a spectrophotometer at a wavelength of 540 nm.

3. Results: Compared with the ribavirin combination group, the CpG OD group and the ribavirin combination group had significantly higher OD values than the CpG ODN group. This result indicates that the synthetic Single-stranded deoxynucleotides have obvious anti-foot-and-mouth disease virus effects, and the combination of synthetic single-stranded deoxynucleotides and ribavirin has a more significant anti-foot-and-mouth disease virus effect. The OD values between the two groups are compared in Table 4. Example 6. Comparison of the effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin on anti-single-stranded RNA Japanese encephalitis virus 1. The isolation of human peripheral blood mononuclear cells is the same as in Example 2.

2. Obtain CpG ODN (6 g/ml) Stimulate human peripheral blood PBMC culture supernatant is the same as in Example 2.

3, synthetic single-stranded deoxynucleotides and synthetic single-stranded deoxynucleotides combined with ribavirin anti-single-stranded strand RNA Japanese encephalitis virus assay

1) Materials: Japanese encephalitis virus was purchased from Changchun Institute of Biological Products. 10% FCS IMDM complete medium: Same as Example 2.

2) Method:

The final concentration of human peripheral blood mononuclear cells was adjusted to ³ x 106/ml with IMDM medium containing 10% calf serum. The cell suspension was added to a 12-well culture plate, and 2 mL per well was added with synthetic single-stranded deoxynucleotides to a final concentration of 6 g/ml. Incubate for 48 hours at 37 ° C, 5 % CO 2 incubator, and collect the supernatant.

CpGODN group, ribavirin group, CpG ODN + ribavirin group, IMDM control group were established. BHK cells (Inner Mongolia Biopharmaceutical Factory) with good growth conditions were inoculated into 96-well culture plates at 1.3×10 ⁴ cells per well, cultured at 37 °C, 5% C0 _{2 for} 24 hours, and CpGODN group was diluted 1:100. CpG ODN stimulated human PBMC culture supernatant, ribavirin group added ribavirin (to a final concentration of 10 μ ιηοΜ), CpGODN + ribavirin group added 1: 100 dilution of synthetic single-stranded deoxynucleotide Stimulated human PBMC culture supernatant and ribavirin (Zhejiang Chengyi Pharmaceutical Co., Ltd.), IMDM group only added the same amount of IMDM. After continuing to culture for 1.5 hours, 100 TCID50 / ml of Japanese encephalitis virus was added, and a normal BHK cell control was set and cultured for 44 hours. Under the microscope, the cytopathic effect (CPE) was observed. 25% of the cytopathic lesions were recorded as "+", 26-50% of the cytopathic lesions were recorded as "++", and 51-75% of the cytopathic lesions were recorded as "+++". 76-100% of cytopathic lesions appear "++++".

3) Results (CPE method): CpG ODN group and CpG OD compared with ribavirin combined application group, CpG

The cytopathic effect of the ODN and ribavirin combination group was significantly lower than that of the CpG ODN group. This result indicates that the synthetic single-stranded deoxynucleotide has a significant anti-Japanese encephalitis virus effect, and the synthetic single-chain deoxygenation. The combination of nucleotides and ribavirin has a more pronounced anti-Japanese encephalitis virus effect. A comparison of cytopathic effects between the two groups is shown in Table 5. The above experimental results show that the artificial single-stranded deoxynucleotide of the present invention has an obvious anti-Japanese encephalitis virus effect, and the synthetic single-stranded deoxynucleotide and ribavirin have a more significant anti-drug application. The role of Japanese encephalitis virus Hepatitis C virus is the same as a single-stranded positive-strand RNA virus like dengue virus and Japanese encephalitis virus. Therefore, synthetic single-stranded deoxynucleotides can also be used in combination with ribavirin. The role of anti-hepatitis C virus. Schedule section

Table 1 Comparison of the effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin on anti-follicular inflammatory virus

Ribavirin group 0.398 ±0.09

Normal VERO cells: 1.625 ± 0.11 Table 2 Comparison of anti-influenza virus effects of synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides combined with ribavirin

Ribavirin group 0.423 ± 0.09

Normal VERO cell control 1.328 ± 0.13 Table 3 In vitro experiments with synthetic anti-influenza virus in combination with synthetic single-stranded deoxynucleotides and synthetic single-chain deoxynucleotides in combination with ribavirin

Model group (negative control) ++++

Normal cell group

Ribavirin group +++ Table 4 Comparison of the effects of synthetic single-stranded deoxynucleotides and synthetic single-stranded deoxynucleotides combined with ribavirin against foot-and-mouth disease virus

IMDM group 0.267 ± 0.05

Ribavirin group 0.561 ± 0.09

Normal BHK cells 1.782±0.14 Table 5 Comparison of the effects of synthetic single-stranded deoxynucleotides and synthetic single-stranded deoxynucleotide N and ribavirin on anti-single-stranded RNA Japanese encephalitis virus

IMDM group + + + +

Ribavirin group + +

Normal BHK cells

Claims

Claim

What is claimed is: 1. A composition for treating or preventing a viral infection and a related disease caused by a viral infection, the composition comprising ribavirin and a synthetic CpG-containing single-stranded deoxynucleotide.

The composition according to claim 1, wherein the artificially synthesized CpG-containing single-stranded deoxynucleotide has a sequence encompassed by one of the following formulas (i) to (v):

(i) (G)n(L)n X1X2CGY1 Y2(M)n (G)n, where X1=A,T,G; X2= A,T; Y1 = A,T;

Y2= A, T, C; L, M=A, T, C, G; n is 0-6;

(ii) (G)n(L)nCG(XY)nCG(M)n(G)n, where X=A,T; Y= A,T; L,M=A,T,C,G; n for

0-6;

(iii) (TCG)n(L)nCG (M)n(G)n, where L, M=A, T, C, G; n is 0-6;

(iv) (TCG)n(L)nXlX2CG(M)n, where X1=A,T,G; X2= A,T _; L,M=A,T,C,G; n is

0-6;

(v) A sequence containing TTCGTCG.

3. The composition of claim 1 wherein said single stranded deoxynucleotide has the sequence set forth in one of SEQ ID NOs: 1-186.

4. The composition of claim 1 or 2, wherein said synthetic single-stranded deoxynucleotide has SEQ ID ΝΟ: 1, 3, 17, 29, 34, 36, 66, 86, 89, 92, 94 Sequence of one of 95, 97, 98, 99, 102, 109, 157, 169, 186.

The composition of any one of claims 1 to 4, wherein the phosphodiester bond of the artificially synthesized single-chain deoxynucleotide is non-vulcanized, partially sulfurized or fully sulfurized.

6. The composition of claim 1 wherein said synthetic single chain deoxynucleotide is chemically modified.

A kit for treating or preventing a viral infection and a related disease caused by a viral infection, comprising a synthetic CpG-containing single-stranded deoxynucleotide and ribavirin, wherein the single-stranded deoxynucleotide has The following formula

Sequence contained in one of (i)-(v)

(i) (G)n(L)n X1X2CGY1 Y2(M)n (G)n, where X1=A,T,G; X2=A,T _; Yl- Α,Τ; Y2= A,T,C ; L, M = A, T, C, G; n is 0-6;

(ii) (G)n(L)nCG(XY)nCG(M)n(G)n, where X=A,T; Y= A,T; L,M=A,T,C,G; n 0-6; (iii) (TCG)n(L)nCG (M)n(G)n, where L, M=A, T, C, G; n is 0-6;

(iv) (TCG)n(L)nXlX2CG(M)n, where X1=A,T,G; X2= A,T _; L,M=A,T,C,G; n is

0-6;

(v) A sequence containing TTCGTCG.

The kit according to claim 7, wherein the single-stranded deoxynucleotide has the sequence shown as one of SEQ ID N0.1 to 186.

The kit according to claim 7 or 8, wherein said single-stranded deoxynucleotide has SEQ ID NO: 1, 3, 17, 29, 34, 36, 66, 86, 89, 92, 94, Sequence of one of 95, 97, 98, 99, 102, 109, 157, 169, 186.

10. The kit of claims 7-9 wherein the phosphodiester bond of the single chain deoxynucleotide is non-sulfided, partially sulfurized, or fully sulfurized.

The kit of claim 7, wherein the single-stranded deoxynucleotide is chemically modified.

12. A method of treating or preventing a viral infection and a related disorder caused by a viral infection, comprising administering to a patient in need of such treatment a therapeutically effective amount of a sequence having a sequence comprising one of the following formulas (i)-(v) Chain deoxynucleotides and ribavirin

(i) (G)n(L)n X1X2CGY1 Y2(M)n (G)n, where X1=A,T,G; X2= A,T _; Y1= A,T; Y2=

A, T, C _; L, M = A, T, C, G; n is 0-6;

(ii) (G)n(L)nCG(XY)nCG(M)n(G)n, where X=A,T; Y= A,T; L,M=A,T,C,G; n for

0-6;

(iii) .(TCG)n(L)nCG (M)n(G)n, where L, M=A, T, C, G; n is 0-6;

(iv) (TCG)n(L)nXlX2CG(M)n, where X1=A, T, G; X2= A, T; L, M=A, T, C, G _; n is

0-6;

(v) A sequence containing TTCGTCG.

13. The method of claim 12, wherein said single stranded deoxynucleotide has the sequence set forth in one of SEQ ID NOs: 186.

The method of claim 12 or 13, wherein said single-stranded deoxynucleotide has SEQ ID NO: 1, 3, 17,

The sequence shown in one of 29, 34, 36, 66, 86, 89, 92, 94, 95, 97, 98, 99, 102, 109, 157, 169, 186.

15. The method of any of claims 12-14, wherein the phosphodiester bond of the single chain deoxynucleotide is non-vulcanized, partially sulfurized, or fully sulfurized.

16. The method of any of claims 12-14, wherein the single stranded deoxynucleotide is chemically modified.

17. The kit of claim 7, wherein the synthetic single-stranded deoxynucleotide and ribavivir The forest can be applied simultaneously, sequentially or in a row.

18. The kit of claim 7, wherein the single-stranded deoxynucleotide of the synthetic single-stranded deoxynucleotide can be used in a single dose of 1 to 5000 mg, and the single application dose of the ribavirin can be 300- 10,000 mg.

19. The kit of claim 7, wherein the synthetic single-stranded deoxynucleotide and ribavirin are administered in a mucosal surface, subcutaneous injection, intramuscular injection, gastrointestinal administration, intraperitoneal administration, and Intravenous injection, etc.

20. The kit of claim 19, wherein said mucosal surface administration is selected from the group consisting of respiratory, digestive tract and genitourinary mucosal administration.

A method of treating or preventing a viral infection and a related disease caused by a viral infection, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of the composition according to any one of claims 1-6.

The method according to claim 12 or 21, wherein the administration mode is mucosal surface administration, subcutaneous injection, intramuscular injection, gastrointestinal administration, intraperitoneal administration, intravenous injection or the like.

The composition according to claim 1 or 7, wherein the virus refers to an RNA virus, a DNA virus and a retrovirus, including influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, and B Hepatitis virus, human immunodeficiency virus and papillomavirus.

24. The method of claim 12, wherein the virus refers to an RNA virus, a DNA virus, and a retrovirus, including but not limited to influenza virus, foot-and-mouth disease virus, dengue virus, Japanese encephalitis virus, hepatitis C virus, B Hepatitis virus, human immunodeficiency virus and papillomavirus.

25. Use of a composition according to any one of claims 1 to 6 for the manufacture of a medicament for the treatment or prevention of a viral infection and a disease associated with a viral infection.