CN100376594C - Compositions and methods to initiate or enhance antibody and major-histocompatibility class i or class ii-restricted t cell responses by using immunomodulatory, non-coding RNA motifs - Google Patents

Compositions and methods to initiate or enhance antibody and major-histocompatibility class i or class ii-restricted t cell responses by using immunomodulatory, non-coding RNA motifs Download PDF

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CN100376594C
CN100376594C CNB038109174A CN03810917A CN100376594C CN 100376594 C CN100376594 C CN 100376594C CN B038109174 A CNB038109174 A CN B038109174A CN 03810917 A CN03810917 A CN 03810917A CN 100376594 C CN100376594 C CN 100376594C
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cell
antigen
motif
dsrna
mouse
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CN1780848A (en
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A·I·博特
王立林
L·德拉玛丽
D·史密斯
B·菲利普斯
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Astral Inc
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Astral Inc
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Abstract

The present application is directed to non-coding RNA motifs that are used in conjunction with an antigen or without an antigen to induce, enhance or modulate an immune response that comprises a B cell and a T cell component.

Description

Induce, strengthen or regulate the immunostimulating double-stranded RNA and the method for immunne response
Invention field
Present invention relates in general to can be used for the motif of induce immune response.Specifically, the application relate to or with under the situation of antigen combination be not used to induce, strengthen or regulate and comprise B cell (antibody) and the optional non-coding RNA motif that comprises the immunne response of T cellular constituent.
Background of invention
A large amount of virus infectiones is relevant with the RNA kind that can not run into usually under standard state, perhaps can cause their generation.Described RNAs is genomic fragment (under the situation of the virus that contains double-stranded RNA s), replicability intermediate or stem-ring-structure, they can be discerned by the congenital immunity acceptor such as Toll-sample acceptor 3 (TLR3), and induce the generation of IFN-I and other solubility media.In addition, confirmed some dsRNA motif already, and can activate the stage that jejune dendritic cell are able to bring into play the effect of special APC as polyI:polyC (pI:pC or pI:C).Although the fact had confirmed already that polyI:polyC and IFN-I can influence the antibody response to proteantigen, the most information restriction of the relevant dsRNA-immunomodulatory motif that produces by the congenital immunity model that is obtained in natural killer cell, scavenger cell and other lack the cell subsets of specific antigens acceptors.Therefore, also do not have to confirm only adaptive immune response is had limited effect, the danger signal that perhaps plays a part to prevent immunological tolerance and instruct the differentiation of specific T cells with motif double-stranded or that other RNA kinds are relevant.In addition, there is the key issue of the relevant dangerous motif of the multiple RNA of potential differentiation influence not obtain answer about whether existing to immunne response.In addition, still fail to determine whether the non-coding RNA motif can promote the inducing of immunne response of I class-restriction during virus infection, also thinking up to date, under most of situation, is to take place after the invalid or productive infection of antigen presenting cell (APC).
During virus infection, the specific T lymphocyte is exposed to the foreign epitope by the MHC molecular display, and the antigen of bone-marrow-derived lymphocyte identification soluble form.Determine the lymphocytic propagation and the differentiation of described adaptive immune response, described immunne response is made up of specific effector cell and memory cell.At the initial period of described immunne response, congenital immunity can be discerned motif relevant with microorganism and the endogenous danger signal of pathology inductive, and this signal can instruct the differentiation subsequently of specific lymphocyte, and the general characteristic of immunne response.Under the situation that lacks danger signal, described T and B cell response strength reduction, and cause immunological tolerance, particularly under medium paramount doses of antigen situation.Proposed already it be differentiate harmless with infect relevant ' danger ' antigenic key mechanism.This mechanism has also shown the Different Strategies of immunity system resolution self and non-autoantigen, thinks in the past and can only determine on antigen-acceptor repertoire level.
Summary of the invention
Adaptive immune response is the identification inductive by T and B cell epitope, and forms by " danger " signal that plays a role through the congenital immunity acceptor.Confirmed already in this application that the motif relevant with non-coding two strands or single stranded RNA provided the key feature of described immunne response, the memory of virus infection, as, the rapid induction of short scorching chemokine expression, the raising and activating, the adjusting of the regulating cell factor, the differentiation of Th1 cell of antigen presenting cell (APC), the isotype conversion and the stimulation of cross-excitation are present in the immunne response of MHC I class-restriction.The application has confirmed that the heterogeneity of the relevant motif of RNA has caused the Different Effects to the immunne response feature.According to the ability of specific RNA-motif blocking-up induction of tolerance and effective histogenic immunity defence during virus infection, confirmed that described RNA kind is effective " danger signal ".The application has disclosed and has utilized selected RNA motif as adjuvant, so that optimize the immunne response to the subunit vaccine.In a word, the relevant motif of the RNA-that produces during virus infection not only has short-term effect to congenital immunity, and gets in touch and reply in early days and the adaptability stage in late period, comprises the activation and the differentiation of antigen-specific b and T cell.
In this application, confirmed except the strand and double-stranded character of RNA that it is the crucial determinative of congenital immunity acceptor identification non-coding RNA motif that described oligonucleotide is formed.In addition, allos RNA motif has effective with different influences to adaptive immunity, most of feature of mediation immunne response during virus infection.At last, confirmed disclosed RNA-motif energy effectively start defense mechanism already, had preventative or therapeutic use aspect communicable disease or the cancer.
The application requires the right of priority of U. S. application serial number of submitting on March 15th, 2,002 60/364,490 and the U.S. Patent application serial number of submitting on September 20th, 2,002 60/412,219, and is received and do this paper reference.
Various embodiments of the present invention comprise:
1. an enhancing is to the method for antigenic immunne response, comprise use the composition formed by double-stranded RNA and with the common applying said compositions of described antigen.
2. method that strengthens or regulate antigenic immunne response when described antigen is Already in the body, comprises and uses the composition of being made up of double-stranded RNA.
3. as the method for paragraph 1, wherein, described RNA is a non-coding RNA.
4. as the method for paragraph 1 or 2, wherein, described double-stranded RNA is made up of poly-VITAMIN B4 and poly-uridylic.
5. as the method for paragraph 1 or 2, wherein, described double-stranded RNA is made up of poly-guanine and poly-cytosine(Cyt) or one of polyinosine and poly-cytosine(Cyt).
6. as the method for paragraph 1 or 2, wherein, described double-stranded RNA is made up of VITAMIN B4 and uridylic.
7. as the method for paragraph 1 or 2, wherein, described double-stranded RNA is made up of guanine and cytosine(Cyt) or inosine and cytosine(Cyt).
8. as the method for paragraph 1, wherein, described method can strengthen Th1 and Tc1 cell response.
9. as the method for paragraph 1, wherein, described method can be induced the Tc1 cell response.
10. as the method for paragraph 1, wherein, described method can strengthen the B cell response.
11. as the method for paragraph 1, wherein, described antigen is used jointly with other antigens.
12. as the method for paragraph 1, wherein, described method can be induced CXC and CC chemokine.
13. as the method for paragraph 12, wherein, described method can be induced MIP-1 α, MIP-1 β, MIP-3 α and IP-10.
14. as the method for paragraph 1, wherein, using of described composition can strengthen T and B cell response by raising and activate CD11b+ monocyte or CD11c+ dendritic cell.
15. as the method for paragraph 1, wherein, the double-stranded RNA composition can enhancing immunity be replied by recovering antigen presenting cell.
16. as the method for paragraph 1, wherein, described antigen presenting cell is special APC.
17. as the method for paragraph 1 or 2, wherein, described double-stranded RNA composition is made up of inosine and cytosine(Cyt), and can induce effectively raising of CD11c+ dendritic cell.
18. as the method for paragraph 1 or 2, wherein, described double-stranded RNA is selected from the RNA composition that contains inosine and cytosine(Cyt) and contains the RNA composition of VITAMIN B4 and uridylic, and described method can be induced, and CD11b+ is monocytic effectively to be raised.
19. as the method for paragraph 15, wherein, described APC is an activated.
20. as the method for paragraph 4, wherein, described antigen is non-infectious antigen, and the T cell of described RNAMHC I class-restriction is by poly-VITAMIN B4 and poly-uridylic RNA composition cross-excitation.
21. as the method for paragraph 4-7, wherein, described RNA composition is by mucosal administration.
22. one kind prevents the method to non-infectious antigenic high-zone tolerance, comprises the double-stranded RNA composition of using described non-infectious antigen together and containing poly-VITAMIN B4 and poly-uridylic or polyinosine and poly-cytosine(Cyt).
23. as the method for paragraph 22, wherein, described non-infectious antigen be use with heavy dose or Already in the body.
24. as the method for paragraph 22, wherein, described dosage is the antigen of dosis tolerata.
25. as the method for paragraph 22, wherein, described method can prevent B cell anergy.
26. as the method for paragraph 1,2 and 22, wherein, described composition and antigen are used by one of following approach: mucosal administration; Respiratory tract is used; Intravenously is used; Subcutaneous administration; And intramuscular administration, with or not compound with various carriers.
27. immunization method, comprise by using antigenic at least one the peptide epitopes antigen loaded that is connected with the Ig main chain to be delivery cell, so that form the Ig-peptide molecule, and use Ig-peptide molecule in the body with the associating of dsRNA motif, wherein, described epi-position is effectively processed and is presented by MHC I approach, has caused effective loading of MHC I quasi-molecule, and be exposed in vivo after the antigen, caused the effective secondary amplification of the T cell of MHC I class-restriction.
28. as the method for paragraph 27, wherein, described antigen is virus.
29. as the method for paragraph 28, wherein, described virus is influenza virus.
30. as the method for paragraph 27, wherein, described peptide-epi-position is reorganization IgG-NP (Kd).
31. as the method for paragraph 27, wherein, described dsRNA is pA:pU.
32. as the method for paragraph 27, wherein, described T cell is a cytotoxic T lymphocyte.
33. as the method for paragraph 27, wherein, be exposed in vivo after the described antigen, the secondary amplification of the T cell of the MHC I class-restriction that causes is better than the recombinant antigen of only using in the Sterile Saline.
34. method of after clinical diagnosis, controlling and treating tumour, comprise by using the relevant t cell epitope antigen loaded of at least one tumour that connects with the Ig main chain to be delivery cell, so that form the IgG-peptide molecule, and use the Ig-peptide molecule of uniting in the body with dsRNA.
35. as the method for paragraph 34, wherein, the t cell epitope that described tumour is relevant is effectively processed and is presented by MHC I approach, has caused effective loading of MHC I quasi-molecule, and has therefore produced MHC I class-peptide complex.
36. as the method for paragraph 34, wherein, described method has caused immunne response and the tumor rejection to the relevant t cell epitope of described tumour.
37. as the method for paragraph 34, wherein, described dsRNA is pA:pU.
38. as the method for paragraph 34, wherein, described IgG-peptide complex and dsRNA treat as Anti-tumor and use repeatedly.
39., wherein, when tumor rejection, produced Tc1 immunity at the tumour associated epitope as the method for paragraph 34 and 35.
40., wherein, when using IgG-peptide and dsRNA, produced Tc2 immunity at the tumour associated epitope as the method for paragraph 34.
41. as the method for paragraph 34, wherein, described method also comprises the effective memory response of inducing identical tumour associated epitope.
42. as the method for paragraph 34, wherein, described method has caused the successive immunity to the tumour cell variant.
43. an enhancing, comprises the described antigen of using and be selected from down the dsRNA associating of group to people or non-human mammal to the method for antigenic antibody response: pA:pU, pI:pC and pC:pG or their mixture.
44. an enhancing, comprises the described antigen of mixture associating of using and be selected from down the single stranded RNA kind of group to people or non-human mammal to the method for antigenic antibody response: pA, pC, pI, pU, p (G, U), p (C, U), p (A, C), and p (I, U), p (C, I), p (A, U), p (A, G), p (A, C, G), p (A, C, U) and p (A, G, U).
45. a composition that is used to strengthen to antigenic immunne response comprises the dsRNA sequence of being made up of poly-VITAMIN B4 and poly-uridylic.
46. as the composition of paragraph 45, wherein, described composition also comprises antigen.
47. as the composition of paragraph 45, wherein, described antigen is Already in the body.
48. as the composition of paragraph 45, wherein, described antigen is to use in carrier that can be medicinal.
49. as the composition of paragraph 45, wherein, described antigen is used in immunoglobulin (Ig).
50. as the composition of paragraph 48, wherein, described can be medicinal be IgG.
51. as the composition of paragraph 45-47, wherein, described antigen is the tumour associated epitope.
52. as the composition of paragraph 45-47, wherein, described antigen is virus.
53. as the composition of paragraph 51, wherein, described antigen is the relevant t cell epitope of tumour.
54. as the composition of paragraph 45-53, wherein, described dsRNA uses with described antigen.
55. as the composition of paragraph 45-53, wherein, described dsRNA and described antigen separate administration.
56. a composition that is used to strengthen to antigenic immunne response comprises the dsRNA sequence of being made up of polyinosine and poly-cytosine(Cyt).
57. as the composition of paragraph 56, wherein, described composition also comprises described antigen.
58. as the composition of paragraph 56, wherein, described antigen is Already in the body.
59. as the composition of paragraph 56, wherein, described antigen is to use in carrier that can be medicinal.
60. as the composition of paragraph 56, wherein, described antigen is used in immunoglobulin (Ig).
61. as the composition of paragraph 59, wherein, described can medicinal carrier be IgG.
62. as the composition of paragraph 56-58, wherein, described antigen is the tumour associated epitope.
63. as the composition of paragraph 56-58, wherein, described antigen is virus.
64. as the composition of paragraph 62, wherein, described antigen is the relevant t cell epitope of tumour.
65. as the composition of paragraph 56-64, wherein, described dsRNA uses with described antigen.
66. as the composition of paragraph 56-64, wherein, described dsRNA and described antigen separate administration.
67. double-stranded (" dsRNA ") is used for strengthening purposes in the medicine of antigenic immunne response in production, comprising: use and described antigen combined described dsRNA to the patient.
68. double-stranded (" dsRNA ") is used for strengthening purposes in the medicine of antigenic immunne response in production, comprising: when described antigen is Already in patient's body, use described dsRNA for described patient.
69. as the purposes of paragraph 67 or 68, wherein, described dsRNA is a non-coding RNA.
70. as the purposes of paragraph 67 or 68, wherein, described double-stranded RNA is made up of poly-VITAMIN B4 and poly-uridylic.
71. as the purposes of paragraph 67 or 68, wherein, described double-stranded RNA is made up of polyinosine and poly-cytosine(Cyt) or poly-guanine and poly-cytosine(Cyt).
72. as the purposes of paragraph 67 or 68, wherein, described double-stranded RNA is made up of VITAMIN B4 and uridylic.
73. as the purposes of paragraph 67 or 68, wherein, described double-stranded RNA is made up of guanine and cytosine(Cyt) or inosine and cytosine(Cyt).
74. as the purposes of paragraph 67-73, wherein, described purposes can strengthen Th1 and/or Tc1 cell response.
75. as the purposes of paragraph 67-73, wherein, described purposes can be induced antigenic Tc1 cell response.
76. as the purposes of paragraph 67-73, wherein, described method can strengthen antigenic B cell response.
77. as the purposes of paragraph 67 or 68, wherein, described antigen is used jointly with other antigens.
78. as the purposes of paragraph 67 or 68, wherein, described method can be induced CXC and CC chemokine.
79. as the purposes of paragraph 67 or 68, wherein, described method can be induced MIP-1 α, MIP-1 β, MIP-3 α and IP-10.
80. as the purposes of paragraph 67-71, wherein, using of described dsRNA can strengthen T and B cell response by raising and activate CD11b+ monocyte or CD11c+ dendritic cell.
81. as the purposes of paragraph 67-71, wherein, the double-stranded RNA composition can enhancing immunity be replied by raising antigen presenting cell.
82. as the purposes of paragraph 81, wherein, described antigen presenting cell is special APC.
83. as the purposes of paragraph 71, wherein, described double-stranded RNA composition comprises inosine and cytosine(Cyt), and can induce effectively raising of CD11c+ dendritic cell.
84. as the purposes of paragraph 67 or 68, wherein, described two strands is selected from the RNA composition that contains inosine and cytosine(Cyt) and contains the RNA composition of VITAMIN B4 and uridylic, and described method can induce that CD11b+ is monocytic effectively to be raised.
85. as the purposes of paragraph 67-70, wherein, described antigen is non-infectious antigen, and the T cell of described RNA MHC I class-restriction is by poly-VITAMIN B4 and poly-uridylic RNA composition cross-excitation.
86. as the purposes of paragraph 67-70, wherein, described composition and antigen are used by one of following approach: mucosal administration; Respiratory tract is used; Intravenously is used; Subcutaneous administration; And intramuscular administration, with or not compound with various carriers.
87.dsRNA be used for preventing purposes in the medicine of non-infectious antigenic high-zone tolerance from comprising the double-stranded RNA composition of using described non-infectious antigen together and containing poly-VITAMIN B4 and poly-uridylic or polyinosine and poly-cytosine(Cyt) in production.
88. as the purposes of paragraph 87, wherein, described non-infectious antigen is that heavy dose is used or Already in the body.
89. as the purposes of paragraph 87, wherein, described dosage is the antigen of dosis tolerata.
90. as the purposes of paragraph 87, wherein, described method can prevent B cell anergy.
91.dsRNA the purposes in producing medicine, comprise by using antigenic at least one the peptide epitopes antigen loaded that is connected with the Ig main chain to be delivery cell, so that form the Ig-peptide molecule, and use Ig-peptide molecule in the body with the associating of dsRNA motif, wherein, described epi-position is effectively processed and is presented by MHC I approach, has caused effective loading of MHC I quasi-molecule, and be exposed in vivo after the antigen, caused the effective secondary amplification of the T cell of MHC I class-restriction.
92. as the purposes of paragraph 91, wherein, described antigen is virus.
93. as the purposes of paragraph 91, wherein, described virus is influenza virus.
94. as the purposes of paragraph 91, wherein, described peptide-epi-position is reorganization IgG-NP (Kd).
95. as the purposes of paragraph 91, wherein, described dsRNA is pA:pU.
96. as the purposes of paragraph 91, wherein, described T cell is a cytotoxic T lymphocyte.
97.dsRNA be used for after clinical diagnosis, controlling and treating the purposes of the medicine of tumour in production, comprise by using the relevant t cell epitope antigen loaded of at least one tumour that connects with the Ig main chain to be delivery cell, so that form the IgG-peptide molecule, and use the Ig-peptide molecule of uniting in the body with dsRNA.
98. as the purposes of paragraph 97, wherein, the t cell epitope that described tumour is relevant is effectively processed and is presented by MHC I approach, has caused effective loading of MHC I quasi-molecule, and has therefore produced MHC I class-peptide complex.
99. as the purposes of paragraph 97, wherein, described method has caused immunne response and the tumor rejection to the relevant t cell epitope of described tumour.
100. as the purposes of paragraph 97, wherein, described dsRNA is pA:pU.
101. as the purposes of paragraph 97, wherein, described Ig-G peptide complex and dsRNA treat as Anti-tumor and use repeatedly.
102., wherein, when tumor rejection, produced Tc1 immunity at the tumour associated epitope as the purposes of paragraph 97.
103., wherein, when using IgG-peptide and dsRNA, produced Tc2 immunity at the tumour associated epitope as the purposes of paragraph 97.
104. as the purposes of paragraph 97, wherein, described method also comprises the effective memory response of inducing identical tumour associated epitope.
105. as the purposes of paragraph 97, wherein, described method has caused the successive immunity to the tumour cell variant.
106.dsRNA be used for strengthening purposes in production, comprise the described antigen of using and be selected from down the dsRNA associating of group to people or non-human mammal: pA:pU, pI:pC and pC:pG in the medicine of antigenic antibody response.
107.dsRNA be used for strengthening purposes in production, comprise the described antigen of mixture associating of using and be selected from down the single stranded RNA kind of group to people or non-human mammal: pA, pC, pI, pU in the medicine of antigenic antibody response, p (G, U), p (C, U), p (A, C), and p (I, U), p (C, I), p (A, U), p (A, G), p (A, C, G), p (A, C, U) and p (A, G, U).
108.dsRNA be used for strengthening purposes in production, comprise the dsRNA sequence of forming by poly-VITAMIN B4 and poly-uridylic to the medicine of antigenic immunne response.
109. as the purposes of paragraph 108, wherein, described composition also comprises antigen.
110. as the purposes of paragraph 108, wherein, described antigen is Already in the body.
111. as the purposes of paragraph 108, wherein, described antigen is to use in carrier that can be medicinal.
Brief description of drawings:
Fig. 1 represents the influence of various synthetic RNA motifs to specific antibody and T cellular immunization;
Fig. 2 represents by the enhancing of specific dsRNA motif to the immunne response of virus antigen;
Fig. 3 represents the influence to congenital immunity and antigen presenting cell of the dsRNA motif determined;
Fig. 4 represents specific dsRNA motif " danger signal " quality;
Fig. 5 represents selected dsRNA motif as effective vaccine adjuvant;
Fig. 6 is the schema of expression dsRNA motif to the influence of immunne response.
Fig. 7 is illustrated in produce during the influenza infection natural, and non-infectious double-stranded RNA has remarkable effect for the specific immune response at proteantigen;
Fig. 8 A represents the various libraries of synthetic RNA motif;
Fig. 8 B represents that different synthetic RNAs has enhancement to B and t cell response at the prototype proteantigen;
Fig. 9 represents the influence to described innate immune responses of the RNA motif selected;
The RNA motif of the uniqueness of the different receptors bind on Figure 10 and the antigen presenting cell;
Figure 11 represents that unique RNA motif can induce the different rise of chemokine;
Figure 12 represents can be by using duplicating of selected synthetic RNA motif control influenza virus;
Figure 13 represents that the synthetic RNA motif pI:pC and the pA:pU that select obviously prevent high-zone tolerance, and described high-zone tolerance is usually with to use a large amount of purifying proteins relevant;
Figure 14 represents the influence to the person monocytic cell of the synthetic RNA motif selected;
Figure 15 A-15B represents the pA:pU of unmarked mistake, rather than the pI:pC of unmarked mistake, can compete the pA:pU that mark crosses and combine with people THP-1 is monocytic;
Figure 16 represents purifying and the fractional separation step of dsRNA;
Figure 17 represents that level part than small molecular weight of the synthetic RNA compound selected has higher biologic activity;
Figure 18 represents it is that pI:pC rather than pA:pU can induce at it self antibody response, has the cross reaction composition at another RNA motif;
After the epi-position that Figure 19 is illustrated in the MHC I class-restriction of IgG mediation is sent and passed, use effectively inducing of selected synthetic RNAs promotion IL-2 and IFN-γ jointly;
Figure 20 represents itself to compare with using described peptide, and the stripped APC by reorganization IgG loads, in that to form MHC I class-peptide complex and generation Tc more effective aspect replying;
Figure 21 is illustrated in when using selected synthetic RNA jointly, passs at the Tc1 that excites the restriction of I class the most effective aspect replying by sending of the epi-position of the I class restriction of IgG mediation;
Figure 22 represents to resist-effectively the exciting to need simultaneously to use to send in the epi-position body of the I class restriction of passing and load APC and carry out suitable guidance of virocyte toxicity T cell by selected synthetic RNA motif by IgG;
Reorganization IgG and selected synthetic dsRNA that Figure 23 represents to have the epi-position of viral I class restriction carry out immunity together, have caused being limited in exciting of the immunne response that infects the virus replication after the invasion and attack;
Figure 24 represents to be used to test the tumor model based on the effectiveness of the molecule of Ig-peptide;
Figure 25 represents with the relevant antigen of tumour APC to be carried out loading in the effective body, activates by selected synthetic RNA motif simultaneously, is enough to effectively control tumor growth and induced tumor and repels, and be to realize that this purpose is necessary;
Figure 26 represents with the relevant antigen of tumour APC to be carried out loading in the effective body, activates by selected synthetic RNA motif simultaneously, can start the effective immunne response at tumor associated antigen;
Figure 27 is illustrated in when using recombination immunoglobulin with tumour associated epitope and the synthetic dsRNA motif of selecting to treat together, and the tumor infiltrating lymphocyte with TXi Baoshouti mark TCR β has obtained the expression of activation tagging CD25;
Figure 28 represents successfully to repel the Tc1 that mouse that tumor treatment crosses produced at the relevant epi-position of the tumour on the therapeutic Ig and replys, and the Tc2 immunity;
Figure 29 represents to pass through the tumor rejection of the treatment inductive success indicated, can effectively prevent the follow-up invasion and attack of identical tumour subsequently, has shown the formation of effective immunological memory;
Figure 30 A-30B is illustrated in the immunity that occurs after the treatment of being indicated that can cause tumor rejection, can prevent the invasion and attack of antigenic variant, and relevant with the overall amplification of cytokine production cell;
Figure 31 A represents: (a) synoptic diagram of natural IgG (light chain-heavy chain heterodimer); (B) antigen (Ag) derived peptide of insertion CDR (complementary determining region) 3,2,1 or framework region; (C) the VH fragment that replaces with antigen or fragment; (D) VH and the CH1 fragment that replaces with antigen or antigen fragment;
Figure 31 B illustrates IgG-peptide and Fc peptide;
Figure 31 C represents the characteristic of the human IgG main chain selected; With
Figure 31 D represents the sequence of described CH, and the synoptic diagram of the construct of expection.
Detailed description of the present invention
Along with the development to the understanding that may act on of congenital immunity, during infected by microbes, exempt from The mechanism that epidemic disease is replied has become the major domain of studying. Recognize soon described congenital immunity Cell has relevant motif or " external source " danger signal of polytype various microorganisms of energy difference Acceptor. After the identification event of described form, the contact between the congenital and adaptive immunity, Have influence on fatefully intensity and the feature of T and B cell response. Described congenital immunity is rapid, But ability to see things in their true light is lower, but can instruct the slower adaptive immunity of development, and comprises more having The effector molecules of effect has obtained a large amount of constituents by somatic mutation. Common adopt congenital and This multimodal recognition strategy of adaptive immunity, with immunity distinguish mode from independently/non-from The master is transformed into the identification of dangerous/non-danger. The relatively poor immunogenicity of purifying protein, by The immune mediator that the microorganism motif carries out induce and described medium (cell factor, chemotactic because of Son and costimulatory molecules) sign of the activity of adaptive immunity is all supported this viewpoint.
As in this application disclosed, adopted rational method that non-coding RNA is described The signal effect of motif has be used to understanding them and control adaptability during virus infections The direct implication of the effect of immunity. In addition, probe into them and be used for immunity inoculation as adjuvant Purposes.
Adopted synthetic RNAs library and dual strategy, adopted adaptive immunity, rather than The impact of congenital immunity as a result of. By this method, be surprised to find except RNA's Outside the double-stranded character, oligonucleotides forms the effect that plays in this respect. Already confirmed based on A:U Motif have and start the Th1 immunity, isotype is transformed into IgG2a (Figure 1A-1C) and intersecting Excite (Fig. 3 A-3E) to than the ability based on the motif higher degree of I:C. The front is limit Fixed I:C motif has caused the T2 that strengthens and B cellular immunity (Figure 1A-1C). With dsRNA Relevant C:G-motif, or the ssRNA motif that mixes have more weak to adaptive immunity Impact is unless use is by the mixture of the ssRNAs of the base composition of complementation. Because described Obtained reproduction under the condition of the functional TLR4 of shortage now, here can get rid of and endogenous toxic material The common approach of plain identification.
Recently, confirmed already that TLR3 was working aspect the pI:pC identification, but, and pA: As if the common identification approach of pU be not showing because of the immune response of inducing by these two kinds of motifs (Figure 1A-1C) that the different feature of work causes. More likely, form in original immunity In the Memory Process of composition, different TLR isotype and/or coreceptors have participated in according to nuclear The character of thuja acid is distinguished the process of RNA motif. A kind of possibility is to be proved and can to identify the palindrome The TLR9 of unmethylated CpG oligodeoxynucleotide motif or the isotype of TLR may be joined With the dsRNA-motif distinguish. But up-to-date evidence does not confirm the participation of TLR9, because of For comparing with unmethylated CpG motif, dsRNA induced different range transcription factor and Costimulatory molecules. Because pI:pC and pA:pU can both induce Gro-beta-T (Fig. 3 A), Such as other media in conjunction with the ability of Th2 cell of having optionally of the chemotactic factor (CF) of CC, Might be responsible for the different Th feature of inducing by described motif.
According to above data, can draw such conclusion: the pA:pU-phase of described new evaluation Close motif and can induce the big measure feature of adaptive immune response, these features are usually only in virus Occur after infecting. T1 replys inducing with proteantigen (OVA of (comprising Th1 and Tc1) And gp140) and the influenza virus of deactivation (Fig. 1-3) obtained record. To proteantigen I class MHC-restriction reply induce (Fig. 3 D, E), show that this RNA motif is enough to APC Be activated to this processing and present the level of machine-processed phase, increased the support cross-excitation as virus The new information of the main mechanism that infects. This shows, may be the relevant dangerous motif of RNA-, Rather than the direct infection of APC determines thin during the picornavirus infection such as influenza virus Cellular toxicity T lymphocyte (CTL) induces. The enhancing of described immune response intensity, Can be interpreted as the raising rapidly and activating of APC (Fig. 3 A-3E). Promote by pA:pU The inducing of T1 immunity, be accompanied by isotype and change, caused the generation of IgG2a antibody (Figure 1B). But, dsRNA can not induce to the isotype of IgA class and change. This and TGF-The inhibition of the β (not shown) of being correlated with shows that the dangerous motif of dsRNA is luring by pro-inflammatory mediator Lead with the downward modulation of Anti-inflammatory mediator and work. Above result and a large amount of, but be not exclusiveness, The dsRNA motif is at the effect kiss that forms aspect the adaptive immunity during such as influenza infection Close.
Because effective dangerous motif can affect the result of immunologic responsiveness and tolerance aspect, grinds Studied carefully dsRNAs and whether can suppress high-zone tolerance to human IgG, it is a kind of already abundant table The immunological unresponsiveness model of levying. Confirmed already that pA:pU and pI:pC were that immunity is anti-The establishment agent that is subjected to (Fig. 4 A-4B), and be not only the conditioning agent that adaptability is replied. This The scope of the feature that dsRNAs has has been enriched in discovery, and has disclosed the assistant of dangerous motif The possibility of agent effect, general at least part of be can by what prevent that immunological unresponsiveness from suppressing to cause Can property. At last, because A and U, rather than I (inosine) base, appear at natural RNA In the kind, described result shows that dsRNA motif and immune response have during virus infections Potential correlation.
PA:pU is as the effectiveness of dangerous motif, is former of control influenza virus by it (Fig. 4 A-4B) of the ability explanation that sexuality is dyed. This feature can be by congenital and adaptability That replys prompts mobilization, and the few DNA motif of unmethylated CpG can change during primary infection The ability of kind immune defense aspect is explaining of highly remembering. Can draw like this by extrapolation Conclusion: congenital immunity have identification external source or an endogenous generation with infect relevant polynucleotides Motif, and correspondingly regulate and control the remarkable ability of described adaptive immunity. Therefore, in system When having antigen, again be exposed to dsRNA may be only can the described immune response of more effective mobilization, The directly removing of prompting antigen.
According to the result that this paper provides, the dsRNA motif is and subunit the epidemic disease of restructuring or deactivation The rational material standed for of the adjuvant of seedling combination. Specifically, lacking in the situation that carrier copies, As if pA:pU might provide some advantage of live vaccine. The application has described and has been used for mucous membrane With the immune complex of system's inoculation, it allows to carry out the common preparation of antigen and dsRNA. As Shown in Fig. 5 A-5D, lung inoculation and cancer immunotherapy with described compound carries out have caused By antibody, the luring of the strong immune response that the T cell of t helper cell and MHCI class-restriction forms Lead.
In a word, by using selection can affect the reasonable side of the RNA motif of adaptive immune response Method has been determined the dangerous motif that unexpected heterogeneity is relevant with new RNA. To described suitable The systematic Study of answering property immune response shows that selected RNA motif can be coordinated various features, These features are memories of natural infection. At last, the application has determined to contain described RNA motif New preparation, it has the potential use for mucous membrane or systemic immunity, and by eliminating Immunological unresponsiveness or tolerance are carried out the potentiality of immunization therapy.
A) materials and methods
1) antigen and immunomodulator
Will be molten available from one group of 18 kinds of strand and the double-stranded synthetic RNAs (referring to table 1) of Sigma Solution is in aseptic PBS. Described RNAs uses or uses separately as merging thing. Ovalbumin (OVA, low endotoxin) is available from Sigma (A7641). B subunit of cholera toxin (CTB) is purchased From Calbiochem (catalog number (Cat.No.) 227039), complete Freund's adjuvant (CFA) is available from DIFCO (order Record numbers 263810), human IgG (hIgG) is available from Sigma (catalog number (Cat.No.) I 4506). Kept Comformational epitope and the restructuring gp140 HIV antigen with trimerizing ability are by importing end Only sudden change is derived by the gp160 envelope protein of bacterial strain IIIB. Antigen is to use Bernard Moss The vaccinia virus vector of doctor (N.I.H.) present is shown in BS-C-1 (ATCC) cell Reach, and by LCA Ago-Gel chromatography (Pharmacia, Piscataway, NJ) purifying. Use is special available from the HIV coating of Fitzgera1d (catalog number (Cat.No.) 20-HG81) The property antibody confirm the identity of gp140 antigen by the Western engram analysis. Influenza virus (bacterium Strain A/WSN/32H1N1) grow at the MDBK cell, and by sucrose gradient centrifugation from upper Purifying in the clear liquid. For inactivation of viruses, under stirring, allow described virion be exposed to shortwave Ultraviolet ray 15 minutes. Confirm described going out by carrying out titration of virus at the mdck cell that allows Live and act on. Obtained in the variable region, to have I-EdHemagglutinin-the derived peptide of-restriction The recombined small-mouse IgG2b of SFERFEIFPKE (IgHA) [Seq.I.D.No.1], and press Method according to the front is carried out purifying.
Table 1
Kind Form
The 1st group of (merging thing 1) the 2nd group of (merging thing 2) the 3rd group of (merging thing 3) the 4th group of the 5th group of (merging thing 4) double-stranded RNA of single stranded RNA (merging thing 5)   p *(A)；p(C)；p(G)；p(I)；p(U)   p(G，U)；p(C，U)；p(A，C)；p(I，U)   p(C，I)；p(A，U)；p(A，G)   p(A，C，G)；p(A，C，U)；p(A，G，U)   pC:pG；pA:pU；pI:pC
*P=" gather "
2) animal
C57BL/6, BALB/c and TLR4-/-the C3H/HeJ female mice, in age in 6-8 week, purchase From Jackson Laboratories (Bar Harbor, MA), and at Alliance Pharmaceutical Corp, stable breeding under the specific pathogen concrete conditions in the establishment of a specific crime. At C57BL/6 and BALB/c Important discovery in the mouse obtains in the C3H/HeJ mouse of induced by endotoxin defectiveness type reaction Reproduction. Female Sprague Dawley rat (250-330 gram) is available from the Taconic farming , and stable breeding under conditions of similarity.
3) immunity inoculation, invasion and attack and virus titer are measured
As indicated above, by intratracheal instillation or aerosolization Mouse and rat is swashed respectively Send out, and for mouse, carry out booster immunization in 2 noses take 2 weeks as the interval. In order to lure Lead high-zone tolerance, by intravenous injection mouse is excited. At last, in order to induce strong immunity Reply, the antigen that is used in emulsification among the CFA carries out subcutaneous inoculation to mouse. Be used for exciting, strengthening Or the antigen consumption of inducing tolerance is: OVA-100 μ g; HIV gp140-10 μ g; HIgG-200 μ g; And the UV-WSN-20 μ g of sucrose purifying. The amount of employed synthetic RNA is 40-50 μ g/ agent are with or without antigen, mix or be not incorporated in short chain lipid (SCL) compound In. Amount/agent of CTB is 10 μ g. Described antigen or send in salt solution and pass is perhaps in preparation The time perfluocarbon (the pure perfluoro-octyl bromide of perflubron[], Liquivent , Alliance Pharmaceutical Corp.) send in and pass, it is compatible with SCL matrix Inert carrier (cumulative volume of instillation or aerosolization is 40-45 μ l).
In order to carry out virus attack, by the nose approach, with sublethal dose (104Tissue culture infective dose 50%-CD50) the WSN virus infections of work with the C57BL/6 of Metofane anesthesia And TLR4-/-C3H/HeJ mouse. After infecting the 5th day, put to death described mouse, taking-up Lung, homogenate and preservation under-70 ℃. By sample and the admissibility MDCK with serial dilution Cell is incubation 48 hours together, uses then chicken red blood cell (available from Animal Technologies) Carry out standard hemagglutination, measure virus titer. The terminal point titre is by three times with interpolation The mensuration estimation, and be expressed as TCID50/ organ.
4) immune complex
Obtain with short chain lipid (SCL) compound (or " immunity of phosphatide as main excipient The technical method of compound ") is spray-drying. Adopted a kind of simpler of the method here Single form. Say simply, in water, phosphatide is carried out homogenate (in order to form liposome or little Group) and with described excipient and active component mix, carry out then spray-drying, more specifically Say: before being about to carry out spray-drying, by mixing two kinds of preparation A and B, preparation contains Water formulation. Preparation A comprises the micelle preparation, wherein, and 0.14g two decoyl phosphatid ylcholine (Avanti Polar Lipids) is dissolved in the hot DI water of 23mL. With 0.0357g CaCl2·2H 2O Be dissolved in the described phospholipid micelle preparation with the 0.714g lactose. Preparation B comprises the 20mg white of an egg The pA:pU of albumen (Sigma) and 4mg (no endotoxin), they are dissolved in the PBS of 5mL In. With the B-191 disk–type spray dryer of standard, under the following conditions to the combination input system Spray-drying is carried out in agent (2mL preparation A and preparation B): inlet temperature=70 ℃, outlet temperature Spend=43 ℃, aspirator=90%, pump=2.2mL/ minute, nitrogen stream=2400L/h. The institute The PL of the compound that obtains: OVA: pApU: CaCl2·2H 2O: the lactose weight ratio is 12: 20: 4:: 3: 61.
5) measure antibody and t cell response
Measure antibody response by ELISA. Saying simply, (is respectively 2 μ g/ml with antigen Gp140, the virus of the sucrose purifying of 8 μ g/ml, the hIgG of 10 μ g/ml or OVA) right The hole is coated with, and with SeaBlock (Pierce, Rockford, IL, catalog number (Cat.No.) 37527) Sealing. At room temperature, with the serial dilutions incubation of serum and BAL fluid at least 2 hours. After washing, the coupling of usefulness alkaline phosphatase (Sigma, catalog number (Cat.No.) A7434) Anti--mouse IgG antibody is developed to analysis, adds then substrate (pNPP, Sigma, order And the automation ELISA reading of SoftMax software is housed by use record N2765), Instrument (Molecular Devices, ThermoMax) is measured.
In order to measure cell response, by allowing organ pass through the nylon Falcon filter screen of 70 μ m (Becton Dickinson, catalog number (Cat.No.) 352350) then, uses the erythrocyte splitting buffer solution (Sigma, catalog number (Cat.No.) R7757) splitting erythrocyte has obtained splenocyte suspension. By using Clostridiopetidase A (Sigma, catalog number (Cat.No.) C9891) digestion lung tissue is passed through Ficoll-Paque then (Amersham Pharmacia, catalog number (Cat.No.) 17-1440-02) gradient centrifugation is correlated with from lung Isolated lymphocytes in the lymphoid tissue. By ELISPOT assay determination t cell response: with 4 The big mouse-anti of μ g/ml-little mouse-anti-IFN γ, IL-2 or IL-4 monoclonal antibody (are respectively BD-PharMingen, catalog number (Cat.No.) 554430, catalog number (Cat.No.) 18161D, catalog number (Cat.No.) 554387) right The cellulose esters flat board of the 45 microns mixing in 96-hole (Millipore, catalog number (Cat.No.) MAHA S4510) Be coated with. Under 37 ℃, in Sterile Saline with after the 10%FCS sealing 1 hour, with 5 * 105Cells/well is added splenocyte suspension, wherein is with or without antigen or peptide. Drench for lung The bar cell, before stimulating with 1: 1 ratio with effector cell and mitomycin-processing The spleen irritation cell of crossing mixes. In order to stimulate, use the gradient amount antigen (OVA, gp140, The WSN virus of hIgG or sucrose purifying) or peptide: the HA SFERFEIFPKE[Seq. of II class-restriction I.D.No.1]; Or the SIINFEKL[Seq.I.D.No.2 of I class-restriction] and in the past The R10K peptide that disclosed HIV V3-derives. After stimulating 72 hours, use biotinylation Big mouse-anti-mouse cytokine antibody (BD-PharMingen) measure, use then chain Mould antibiotin-HRP (BioSource Int., Camarillo, CA) and insoluble AEC Substrate develops to mensuration. Multi parameter analysis software (Image Pro, Media are equipped with in utilization Cybernetics) automation imaging system (Navitar/Micromate) is measured described The result.
6) mensuration of chemokine gene expression
By following DNA array technique, with synthetic RNA or control treatment 1 day before measuring The expression of chemotactic factor (CF) in the mouse lung: usefulness RNeasy kit (Qiagen, Valencia, CA) from lung, separate total RNA. By DNaseI (Stratagene, the San with no RNase Diego, CA) process, be further purified RNAs. By using available from SuperArray Inc. The Nonrad-GEArray kit of (Bethesda, MD) carries out the DNA array. Simply Say, utilize the MMLV reverse transcriptase, use the dNTP mixture that contains biotin-16-dUTP Synthetic cDNA probe. Under 68 ℃, allow described GEArray film prehybridization 1-2 hour. Hybridization To be undertaken by the cDNA incubation that described film is crossed with biotin-mark. Hybridized Film washs in 2 * SSC-1%SDS 2 times, and washs 2 in 0.1 * SSC-0.5%SDS Inferior. Further use alkaline phosphatase-coupling streptavidin (BioSource Int., Camarillo, CA) the described film of incubation, and finally develop with the CDP-Star chemical luminous substrate. With Gel-Pro software (Media Cybernetics, Silver Springs, MD) is housed Image-Pro analytical system measured signal intensity.
7) flow cytometry
By carrying out as stated above collagenase digesting and Ficoll gradient centrifugation, preparation in advance Pneumonocyte suspension with synthetic RNA or 1 day mouse of saline treatment. With described cell again Be suspended in and contain 1% (v: v) mice serum (Sigma. catalog number (Cat.No.) M5905) and 1% (w: V) bovine serum albumin(BSA), the salt of the phosphoric acid buffer of level part V (Sigma, catalog number (Cat.No.) A3059) In the water, and the big mouse-anti of crossing with the PE-mark-mouse CD11b (PharMingen, catalogue Number 01715A), the hamster that the PE-mark is crossed anti--mouse CD11c (PharMingen, catalog number (Cat.No.) 09705A) or suitable PE-mark the isotype contrast (PharMingen, the catalog number (Cat.No.) 11125A that cross Or 11185A), with 1 μ g antibody/106The concentration of cell dyeed 40 minutes on ice. With Becton Dickinson FacsCalibur instrument carries out described analysis. Get rid of with iodate third ingot The cell of not surviving.
8) magnetic separation and adoptive transfer
By the magnetic bead of utilization with big mouse-anti-little mouse-anti-CD11c antibody (Miltenyi Biotech) coupling, from the spleen of BALB/c mouse, separate such as CD11c+Dendritic cells special APC. Say simply, with 107The density of cell/ml with single-cell suspension liquid Eddy diffusion at MACS In the buffer solution (BSA and EDTA), on ice with magnetic bead incubation 15 ', washing and logical Cross the magnetic post. Before wash-out, wash described post three times, carry out continuously subsequently 2 washings, and And, be with or without 50 μ g/ml RNA motifs, or 5ng/ml rIL-12 (Biosource Int., Camarillo, CA) condition under, with the external pulse of the IgHA of 100 μ g/ml Night. In addition, in advance with big mouse-anti-mouse CD40 monoclonal antibody (BD-PharMingen) On the coated hole, described cell is incubated overnight with IgHA. Wash described cell, heavy Newly be suspended in the Sterile Saline of balance, and arrive not contacted by the hypodermic injection adoptive transfer The BALB/c mouse (2.5 * 10 of antigen5The APC/ mouse) in the body. At the 14th day, pass through IL-2 The described t cell response of ELISPOT assay determination is then according to the method described above with II class HA-limit The peptide of system stimulates.
9) statistical analysis
The intensity of utilizing t-test relative immunity to reply supposes that the normal distribution of described value is with equal Variance.
B. result
1) system definition of the RNA motif of adjusting immune response
During virus infections, produced instantaneous, unusual RNA kind, and played " danger By inches " the effect of signal. Therefore, infer that multiple different RNA motif is by the congenital immunity cell Identification, and far-reaching the adaptive immune response of regulating. In order to explain this in system-based Plant hypothesis, screened synthetic strand and double-stranded RNA motif library and regulated executing by respiratory tract With the specific IgG of proteantigen (OVA) ability of replying.
In order to simplify this process, described screening is organized into two-wheeled: (1) first round relates to RNA The merging thing (table 1) of kind; With, (2) second take turns analysis has described immune response Composition in the merging thing of big impact.
Double-stranded RNA of the present invention (dsRNA) or single stranded RNA (ssRNA) are by the Sigma public affairs Department prepares in accordance with the following methods: ssRNA: polynucleotides (polyA, polyU) are to utilize Nucleotides and polynucleotides-phosphorylase, by enzymatic method preparation, do not come from animal Material enter its preparation process. DsRNA: allow polyadenylic acid (polyA, pA) and poly-uridine Acid (polyU, pU) annealing. Generally, for dsRNA, dsRNA of the present invention and ssRNA Be homopolymers, as one man consist of one by independent a kind of base or nucleotides (for example, adenine) The bar chain as one man consists of another chain by his complement. For ssRNA, described list Chain is to be made of equably identical nucleotides. But, use is (right by the nucleotides that mixes In dsRNA, and complement) dsRNA or the ssRNA composition that form belong to this Bright scope. DsRNA of the present invention and ssRNA composition be by base/nucleotides adenine (A), Guanine (G), cytimidine (C), uracil (U) and inosine (1) form. Table I and Fig. 8 A Shown RNA composition has illustrated the various RNA compositions that are used for these embodiment. The present invention The RNA composition be according to the preparation of the method for example 27 and purifying.
The length that is used for various RNA chains of the present invention is generally 100-2000 base-pair, But, its length can be 1-20,20-40, and 40-60,60-80,80-100,1-100, 100-200,200-300,300-400,400-500,500-600,600-700,800-900, 1000-1100,1100-1200,1200-1300,1300-1400,1400-1500, 1500-1600,1600-1700,1700-1800,1800-1900,1900-2000,2000-2100,2100-2200,2300-2400,2400-2500,2500-3000,3000-4000, 4000-5000,5000-10,000 base-pair, and surpass 10,000 base-pairs, And/or their mixture.
The various merging things of embodiment 1:RNA motif are to the influence at the antibody response of standard antigen (OVA)
Merging the influence of thing (table 1) by respiratory tract with the various RNA of C57B1/6 mouse assay that OVA carries out common immunity to adaptive immunity.As described in " material and method " part, described antibody response is (the n=4/ group) represented with the mean value of IgG terminal point titre ± SEM form.In contrast, use the OVA among the aseptic PBS respectively, had OVA (CTB) and the independent PBS of Toxins,exo-, cholera subunit B.Shown in Figure 1A, be equivalent to the merging thing that antagonist is replied the dsRNA with maximum effect, have obvious enhanced specificity immunity.In addition, the merging thing of strand kind complimentary to one another has reproduced this enhancement.
This shows the residue character of RNA and secondary structure " danger " ability of motif effect that all determining them to play, and specific b cells is replied exert an influence.
Embodiment 2: various one dsRNA motifs are to inducing the influence at the antibody response of OVA
Experimentize according to the method disclosed in front example and " material and method " part, but, we do not use RNAs to merge thing, and are to use one dsRNAs.The result in the mode identical with Figure 1A shown in Figure 1B.Described result represents two independently experiments.Illustration: the ratio of average IgG2a and IgG1 titre and OVA.Order from left to right is similar with master map: PBS OVA, CTB OVA, pC:pG OVA, pI:pC OVA and pA:pU OVA.Take turns in the screening at second shown in Figure 1B, confirmed that two types motif is formed double-stranded RNA, i.e. pA:pU and pI:pC, they have obvious influence to the generation of replying at special antigenic IgG in the C57BL/6 mouse body.To endotoxic reply impaired TLR4-/-obtained the similar results (not shown) in the C3H/HeJ mouse.
Therefore, one RNA motif shows that in intensity and characteristic aspect the influence that antagonist is replied has different abilities.
Embodiment 3: by the intensity and the feature of OVA and various one dsRNA motif inductive t cell responses
Analyze the result who obtains by ELISPOT and be expressed as the IFN-γ of each spleen and mean value ± SEM (n=4/ group) that the IL-4 spot forms colony (SFC) quantity.Shown in Fig. 1 C, pA:pU and pI:pC (40 μ g are referring to " material and method ") reply specific T-cells has enhancement.It is shocking, the feature of the t cell response of specific antigen is depended on the character of RNA residue, this shows that immunity system can offer an explanation the relevant dangerous motif of RNA.Contain A and U residue, rather than the RNA motif of I and C, in C57BL/6 mouse body, can instruct produce IFN-γ-the differentiation of Th1 cell (Fig. 1 C).This result has embodied the different inducing action of IgG isotype, coincide with described Th feature (Figure 1B-INSET).
In a word, different dsRNA motif intensity that t helper cell is replied has different influences with feature.
Embodiment 4: the dsRNA motif of determining is to the influence at the proteic antibody response of virus antigen HIV reorganization gp140
Shown in Fig. 2 A, with by the respiratory tract list with antigen or with of the influence of the immune C57BL/6 mouse assay of antigen and pA:pU (40 μ g are referring to " material and method ") to the segmental antibody response of HIVgp140 (10 μ g, material and method ").The result is expressed as the mean value ± SEM (n=3/ group) of IgG terminal point titre.
Therefore, by using new dsRNA motif to strengthen antibody response to virus antigen with potential practical value.
Embodiment 5: the dsRNA motif of determining is to the influenza virus at complete UV-deactivation, the influence of the antibody response of virus strain A/WSN/32H1N1 (UV-WSN)
The same with embodiment 4, after carrying out the mucosal immunity inoculation, measure influenza virus-specific IgG antibodies with the WSN virus (UV-WSN) (20 μ g are referring to " material and method ") self of UV-deactivation or with dsRNA motif (50 μ g).In contrast, used with the antibody response (n=4/ group) after the identical strains of influenza viruses infection.Result's mean value ± SEM form with IgG terminal point titre in Fig. 2 B is represented.
Therefore, by using new dsRNA motif to strengthen antibody response to the virus antigen of the microorganism form of complete deactivation.
Embodiment 6: by using the t cell response of definite dsRNA motif enhancing to the influenza virus of complete-deactivation jointly
By being carried out ELISPOT, splenocyte analyzed and researched to the t cell response of complete influenza virus, and, after the deduction background, be expressed as IFN-γ, IL-4 and IL-2 SFC (mean value ± SEM, n=4/ group).To compare (referring to Fig. 2 C) at the t cell response of antigen and pA:pU and with replying after inoculation of antigen autoimmunization or the influenza infection.
Therefore, as what in embodiment 4-6, confirmed, the influence exogenous antigen that the dsRNA antagonist is replied, promptly the influenza virus of HIV envelope protein (reorganization gp140) and complete-deactivation obtained respectively confirmation (Fig. 2 A, B).In fact, be pA:pU, rather than pI:pC, with specific antibody to the titre of influenza virus return to by infection induced similar level (Fig. 2 B).In addition, and it is apparent that when carrying out immunization with the virus of deactivation, pA:pU returns to t cell response the level (Fig. 2 C) that is reached by natural infection.Therefore, different RNA motifs have produced the wide influence of former the unknown to T and B cell response.
2) relevant with dsRNA motif can be regulated raising and activating of special antigen presenting cell
Someone supposes the dangerous motif relevant with dsRNA, can be by the composition remote effect t cell response of congenital immunity as pA:pU and pI:pC.In order to verify this hypothesis, after administering RNA s, in the lung Lymphoid tissue, measured the expression of chemokine gene.
Embodiment 7:dsRNA motif raises the part that chemokine gene is expressed
By after the respiratory tract therapeutic 1 day, measured the part rise that the dsRNA motif is expressed chemokine gene by DNA array technique (referring to material and method " mensuration that chemokine gene is expressed ").The result is expressed as the increase multiple of the expression level of measuring relatively in the lung tissue of untreated mouse.By dsRNA inductive chemokine expression pattern with by LPS inductive expression pattern different.Can represent (Fig. 3 A) with the profile of successive and interruption optionally in conjunction with the chemokine of the acceptor on Th1 and the Th2 cell respectively.
Described DNA array technique shows, IP-10, MIG, MIP-1 α, MIP-1 β and MCP-1 have been subjected to the inducing by force of pA:pU and pI:pC (referring to Fig. 3 A).But, have only pI:pC to induce and have the RANTES of combination by the ability of the acceptor of Th2 cell selective ground expression, the expression of MCP-3 and CC chemokine.LPS has induced different chemokine expressions: raise CXC chemokine MIG and NIP-1 α, and CC chemokine TCA-3 (referring to Fig. 3 A).Therefore, as what do not reckon with in the past, the complex characteristic of chemokine is the dsRNA motif inductive by determining.
Embodiment 8: raise special APC in the mouse lung of handling with dsRNA
After handling 1 day, be evaluated at raising of APC special in the mouse lung with the dsRNA processing by facs analysis.In Fig. 3 B, described result is expressed as CD11c +And CD11b +Cell accounts for the per-cent (n=4/ group) of cell colony total in the interstitial tissue of lung.CD11b +(monocyte) raised by pA:pU and pI:pC, consistent with the rise of chemokine expression (referring to embodiment 7).On the contrary, have only pI:pC can effectively induce CD11c +Raising of dendritic cell.In a word, the dsRNA motif can be raised APC in site of administration.
Embodiment 9: activate special APC (dendritic cell) by the dsRNA motif
Confirm of the activation of dsRNA motif by the following method: with antigen and the stripped pulse CD11c of dsRNA to special APC +Cell enters not contacted antigenic BALB/c mouse by the adoptive transfer experiment then, and measures t cell response (Fig. 3 C).In contrast, used the APC of antigen pulse respectively, or the antigen loaded cells that stimulates altogether with rIL-12 and anti-CD 40 mAb.In the result shown in Fig. 3 C, be expressed as by the IL-2 SFC quantity in the spleen of ELISPOT analytical estimating.
Therefore, except described recruitment, the dsRNA motif can activate APC.
Embodiment 10: stimulate the T cell of the antiviral antigenic MHC I class of cross-excitation-restriction in BALB/c mouse by the dsRNA motif.
Analyze by ELISPOT, in the BALB/c mouse body that HIVgp140 antigen (10 μ g) and pA:pU with the production of reorganization-engineering handled together, study the cross-excitation that stimulates by the dsRNA motif and (be meant the specific occasion, at this moment, do not having under the situation about infecting, APC has obtained to excite the ability of the T cell of I class restriction), utilize the stimulated in vitro (referring to " material and method ") of being undertaken by the related peptides of MHC I class-restriction.In contrast, used the gp140 antigen of dosage coupling.In Fig. 3 D, described result is expressed as the mean value ± SEM (n=4/ group) of IFN-γ and IL-4 SFC quantity/spleen.
In a word, the dsRNA motif helps inducing non-infectious antigenic MHC I class-restriction with potential practical use.
Embodiment 11: in the C57BL/6 mouse, stimulate by the dsRNA motif, cross-excitation is at the T cell of MHC I class-restriction of OVA.
Analyze by ELISPOT, in the C57BL/6 mouse body of handling together with complete OVA and pA:pU, studied the cross-excitation that stimulates by the dsRNA motif, the stimulated in vitro that the peptide of utilization by MHC I class-restriction carries out (referring to " material and method ").In contrast, the OVA antigen or the aseptic PBS of dosage coupling have been used.In Fig. 3 E, described result is expressed as the mean value ± SEM (n=4/ group) of IFN-γ and IL-4 SFC quantity/spleen.
In a word, the dsRNA motif helps inducing non-infectious antigenic MHC I class-restriction with potential practical use.
After by mucosal administration pA:pU and pI:pC,, show to have promoted CD11b to the facs analysis that the interstitial lung cell carries out +Monocytic raising, and under second kind of situation, promoted CD11c +Dendritic cell are raised (Fig. 3 B).In addition, external incubation is from the CD11c of contacted antigenic mouse not +DC and antigen and pA:pU, and on less degree with pI:pC Wen Yong, caused their activation because subsequently with the cell adoptive transfer of antigen pulse in the BALB/c acceptor, induced the T cellular immunization (Fig. 3 C) of enhanced II class-restriction.Measured similar enhancement by the APC incubation that carries out with anti-CD 40 antibodies or IL-12.At last, when carrying out mucosal vaccination with Recombinant HIV envelope protein (gp140) or OVA, pA:pU has strong CD8 +T cell-promoter action (Fig. 3 D-E), the ELISPOT that carries out as the peptide of using the MHC I class-restriction of crossing from the sign of HIV envelope protein and OVA respectively analyzes indicated.Therefore, dsRNA has the induction of the special APC ability to the stage compatible with the cross-excitation of the T cell of MHC I class-restriction.Such immunne response only can run in the occasion of virus infection usually.Use the dsRNA motif of determining, can eliminate the needs to the living vaccine carrier, it is relevant with the side effect that produces because carrier duplicates.In a word, above result has shown the remarkably influenced of RNA motif to the factor of congenital immunity, and the latter has the ability of regulation and control adaptive immune response.
3) the dsRNA motif can be blocked inducing of high-zone tolerance and induce preventative resisting-virus immunity
The a bad actor participates in differentiating harmless antigen and the antigen relevant with course of infection.Under heavy dose of situation, non-infectious purifying protein antigen can be induced anergy or immunological tolerance.Acquisition is " immunity is ignored " and " immunological tolerance " to the main path of self or harmless antigenic tolerance.Under first kind of occasion, because spatial separation, antigen can not contact APC.Under second kind of occasion, the antigen of contact APC is processed by internalization, and resulting epi-position is presented common the stimulation under the more weak situation.Net result is to have induced immune reactionless or tolerance on the T cell levels.Under infection or immunostimulation occasion, there is the mechanism that suppresses " immunity is ignored " and " tolerance ".Described mechanism is by the new migration model of inducing APC and activates costimulatory molecules and carry out with short scorching chemokine and cytokine expression.Strong immune response consequently, rather than any antigenicly ignored or tolerate what immunity system exposed under the occasion that the existence by " a bad actor " limits.As a kind of example, tumor associated antigen is ignored by immunoeffectors usually or is occurred with the tolerance form.Recovery has practical significance to the competent mode of described antigenic immunity in anticancer therapy.In order to check the danger signal competence of pA:pU and pI:pC motif, used the tolerance model that obtains by intravenous inoculation hIgG.
Embodiment 12: in the mouse body of injecting with human IgG, the dsRNA motif can prevent high-zone tolerance.
At first, with the hIgG self (filled symbols) of standard tolerance dose (200 μ g) or with pI:pC or pA:pU (40-50 μ g) (hollow symbol; Referring to Fig. 4 A) mouse is carried out intravenous injection, use the hIgG that is emulsified among the CFA of immunizing dose (50 μ g) to carry out subcutaneous booster immunization subsequently.After booster immunization, by the antibody titers of ELISA with the anti-hIgG of various measuring spaces.In contrast, use the hIgG that is present among the CFA that mouse is carried out immunity, and represent maximum titre (discontinuous lines).In Fig. 4 A, described result is expressed as the mean value ± SEM (n=5/ group) of terminal point titre.
Above result shows, co-inoculation pA:pU or pI:pC and be present in hIgG in the salt solution can suppress inducing of B cell anergy, as with being present in hIgG among the CFA (Fig. 4 A) that antibody titers confirmed after carrying out booster immunization.The motif that described dsRNA is relevant can be induced stronger primary response, and recovers the antigenic secondary reaction of its prototype is answered.Similarly, the assessment of T cell characteristic shows, by using dsRNA jointly, the generation that part has been recovered IL-4 and IL-2.
Embodiment 13:dsRNA motif has the different ability of mobilization at the immune defense of influenza infection
By inference, dangerous motif has the ability of the prevention mechanism of the immune defense of prompting mobilization.Therefore, test pA:pU and pI:pC and whether the primary infection of influenza virus has been had any influence.
With before the influenza infection lung of sublethal dose and 1 day afterwards, by respiratory tract pI:pC, pA:pU or brine treatment mouse.At the 5th day, estimated the virus titer in the lung tissue, and be expressed as total infectious unit/organ (mean value ± SEM; The n=6/ group; The result is illustrated in C 3H/HeJ TLR-4-/-and the competence mouse in twice independent studies of carrying out).
Shown in Fig. 4 B, the result shows, has been subjected to the infection of the influenza virus of sublethal dose with the mouse of RNA motif processing by respiratory tract.After infecting 5 days, the virus titer of lung is carried out quantitatively.With C57BL/6 and TLR4-/-C 3The H/HeJ mouse has obtained the similar results (not shown).Obviously, effective reduction that described dsRNAs can effective coordination Pneumovirinae titre.It is shocking that aspect control Pneumovirinae titre, pA:pU is obviously more effective than pI:pC, this has further given prominence to the ability that described immunity system is differentiated the relevant dangerous motif of dsRNA.Therefore, under the situation that lacks immunological memory, the dsRNA motif can be mobilized the effective primary response at virus infection.
4) by sealing the immunity that strengthens proteantigen jointly with the dangerous motif of RNA
Because to the immunne response of subunit vaccine of preparation not, and in general, very weak to the antigenic immunne response of purifying protein, checked in vivo, in identical microstructure, allow APC be exposed to the dangerous motif of antigen and dsRNA simultaneously and whether can cause more favourable result.For this reason, with the pA:pU in prototype antigen (OVA) and the short chain composite of lipid (" SCL ") or pI:pC (referring to " material and method ") prepare jointly with biocompatibility and immunology inertia phosphatide (for example, two decoyl phosphatidylcholines) and as the lactose of vehicle.Be formulated in OVA+ pA:pU in the short chain composite of lipid or pI:pC send be delivered in the C57BL/6 mouse breathing road after, measure antibody response, this reply obvious than with the antibody response of the mouse of the antigen of the not preparation in the salt solution or the antigen inoculation in the SCL mixture that lacks the dsRNA motif, prepared (Fig. 5 A) by force.
Embodiment 14: model antigen (OVA) self or the short chain composite of lipid that loads with the dsRNA motif.
Prepared form by short-chain phospholipid and with model antigen (OVA) self or the short chain composite of lipid that loads with the dsRNA motif, and in the C57BL/6 mouse, test, shown in Fig. 5 A.Measure described antibody response by ELISA, and be expressed as the interior immunization of the tracheae mean value ± SEM of 2 all IgG terminal point titres (n=4/ group afterwards; Data representation is independently measured for twice).In contrast, OVA that is present among the PBS and the OVA for preparing jointly with the CTB (cholera toxin B) that is present in the short chain composite of lipid (two decoyl phosphatidylcholines) have been used.Described result shows, can prepare the molecular complex that comprises antigen and dsRNA of the immunological characteristic that has kept described RNA motif, and have practical value.
Embodiment 15: after the OVA immunization of using with the common preparation of dsRNA motif, in C57BL/6 mouse body to part (lung) and system's (spleen) t cell response of the dominance OVA peptide of complete OVA antigen or I class-restriction.
Fig. 5 B is illustrated in the C57BL/6 mouse part (lung) and system's (spleen) t cell response to the dominance OVA peptide of complete OVA antigen or I class-restriction, and it is to measure with the OVA mice immunized that is present in the short chain composite of lipid (two decoyl phosphatidylcholines) that is with or without pA:pU.Described analysis is undertaken by ELISPOT, and the result is expressed as IFN-γ SFC/ organ (mean value ± SEM; The n=4/ group).
What is interesting is that under the situation of the common preparation of short chain composite of lipid, CTB only has limited adjuvant effect.Coincide with former result, shown in Fig. 5 B, only inducing of T1 immunity measured by the pA:pU particle, rather than measured by pI:pC, and the latter only shows the enhancing (not shown) of T2 immunity.In addition, the common antigenic t cell response of preparation of pA:pU (referring to " material and method ") is shown important local composition (Fig. 5 B).At last, utilize the SIINFEKL[Seq.I.D.No.2 of clear and definite already I class-restriction] peptide, confirmation pA:pU has kept with the SCL mixture and has united the ability (Fig. 5 B) that promotes cross-excitation.
Embodiment 16:Sprague-Dawley rat carries out the system and the local antibody response of mucosal vaccination to using the SC-composite of lipid that loads by the model antigen (OVA) with the common preparation of dsRNA motif.
With rat being inoculated with the composite of lipid of OVA and the common preparation of dsRNA.In contrast, used respectively to lack antigenic SCL mixture, with OVA load but lack the SCL mixture of dsRNA motif and be present in the OVA of the dosage coupling amount in the salt solution.Described result is expressed as the OVA-specific antibody terminal point titre (mean value ± SEM that measured respectively by ELISA at the 35th day in serum (Fig. 5 C) and bronchial perfusate (Fig. 5 D); The n=4/ group).
For the Sprague-Dawley rat, carry out aerosolization with the SCL mixture that has loaded OVA and pA:pU or pI:pC, measured the similar enhancing (Fig. 5 C) of antibody response.With the SC-composite of lipid that lacks dsRNAs or be present in OVA in the salt solution, obtained lower titre.Similar characteristics has been found in the analysis of mucoantibody titre (Fig. 5 D).
Therefore, prepare RNA relevant dangerous motif and proteantigen jointly, preserved the immunomodulatory properties of RNA motif, and caused the remarkable enhancing of local and systemic characteristic immunne response by using new spray drying technology.
Embodiment 17: the non-dsRNA of duplicating motif plays a part the master switch of adaptability (B and T cell) immunne response.
The antigen that lacks dangerous motif such as dsRNA is weak immunogenic, if perhaps heavy dose provides, and possible inducing immune tolerance.But, the dsRNA motif can change immunity system and discover described antigenic mode: what replace weak reactivity or tolerance is that described motif indication adaptability (T and B cell) immunity produces strong reaction to simultaneous antigen, and prevents or the blocking immunity tolerance.Therefore, the congenital immunity of utilizing the dsRNA motif to discern plays a part the master switch (Fig. 7) of adaptability B and T cellular immunization.
Embodiment 18. naturally occurring dsRNA can get in touch congenital and adaptive immune response.Embodiment 18 is illustrated in produce during the influenza infection natural, and non-infectious double-stranded RNA has remarkably influenced to the specific immune response at proteantigen.
With WSN influenza virus (10 8TCID 50/ 1 * 10 9Cell) infect the admissibility mdck cell, and after 24 hours, gather in the crops described cell, wash, and (Qiagen, Valencia CA) extract total RNA with the RNA separating kit.(Stratagene, San Diego CA) handle, and are further purified described RNA by the DNAseI with no RNAse.Then, by under 37 ℃, with the S1 nuclease of 5 μ (Ambion, Inc., Austin, TX)/single stranded RNA that μ g RNA incubation was removed in the sample in 30 minutes.Before described digestion and afterwards, by the described RNA of gel electrophoresis analysis.Confirm the existence of infection characterization of the dsRNA of purifying by the titration of standard influenza virus.In contrast, used from 10 9The individual mdck cell that did not infect with the material of similar approach purifying and processing.By spectrophotometry (A 260nm) measure nucleic acid concentration, and analyze the endotoxic shortage of confirmation by Limulus.Used the dsRNA and the contrast RNA of purifying respectively, perhaps as with the mixture (antigen of the RNA of 25 μ g and 2 μ g is present among the aseptic PBS of 25ml) of gp140 recombinant antigen.
After confirm lacking infectivity, dsRNA or the contrast RNA of 40 μ g mixed with the antigen (gp140HIV coating) of the reorganization brachymemma of 40 μ g, and instil by nasal cavity and to use (n=3/ group) to BALB/c mouse.Other contrasts are with the animal that is present in 40 μ g gp140 albumen (n=3/ group) inoculation in the salt solution.After exciting for 2 weeks, mouse is carried out booster immunization one time.Gather blood at booster immunization after 2 weeks, preparation serum, and by the antibody response of ELISA mensuration at gp140.Say that simply (2 μ g/ml gp140) wraps quilt to the hole with antigen, and seal with SeaBlock (Pierce, Rockford, IL, catalog number (Cat.No.) 37527).At room temperature, with the serial dilutions incubation of serum and bronchial perfusate at least 2 hours.After the washing, with with alkaline phosphatase (Sigma, catalog number (Cat.No.) A7434) link coupled anti--mouse IgG antibody develops to described mensuration, add substrate (pNPP then, Sigma, and utilize the automatization microtiter plate readout instrument that SoftMax software is housed (Molecular Devices ThermoMax) measures catalog number (Cat.No.) N2765).
In the picture A of Fig. 7, show the General Principle of described experiment.In Fig. 7, picture B shows for complete IgG, in the absorption of measuring after developing, is equivalent to various serum dilutions.In Fig. 7, picture B represents that for IgG2a and IgG1 antibody isotype the ratio with 1/50 is carried out the absorption of serum dilution.
Generally, the data of the picture A-B among Fig. 7, expression has unexpected enhancement from natural, the non-infectious dsRNA of the mdck cell of influenza virus-infection for replying at the antigenic adaptability of prototype.IgG1 and IgG2a antibody response all are enhanced, and show to have induced auxiliary 1 and 2 reactions of strong T.
The selected RNA motif of embodiment 19. is to the influence of innate immune responses: the allos motif.
Present embodiment shows that unexpectedly different synthetic RNA motifs have different influences to the adaptability specific immune response at proteantigen.
Fig. 8 A represents the library widely of synthetic RNA motif, these libraries is divided into various merging things, and is used for following pair of titre screening process:
(A) merge thing with RNA described mouse is carried out immunity in the tracheae, 2 weeks at interval subsequently, instil by nasal cavity and to carry out booster immunization 2 times.Be expressed as the mean value ± SEM (n=4/ group) of IgG terminal point titre by the antibody response (Fig. 8 B) of ELISA mensuration.In contrast, use the OVA of the dosage coupling that is present among the aseptic PBS respectively, OVA and cholera toxin subunit b (CTB) and independent PBS.Say that simply (OVA of 10 μ g/ml) wrap quilt to the hole with antigen, and seal with SeaBlock (Pierce, Rockford, IL, catalog number (Cat.No.) 37527).At room temperature, with the serial dilutions incubation of serum and bronchial perfusate at least 2 hours.After washing, with with alkaline phosphatase (Sigma, catalog number (Cat.No.) A7434) bonded anti--mouse IgG antibody develops to described mensuration, add substrate (pNPP then, Sigma, and utilize the automatization microtiter plate readout instrument that SoftMax software is housed (Molecular Devices ThermoMax) measures catalog number (Cat.No.) N2765).
(B) various dsRNA motifs are to inducing the influence at the antibody response of OVA: the result is shown in Fig. 8 C.The result represents two independent experiments.Illustration: the ratio of average IgG2a and IgG1 titre and OVA.For this reason, used vitamin H-link coupled to resist-mouse IgG1 and IgG2a antibody, cultivated with streptavidin-AKP conjugate then.Order from left to right is similar with main picture among Fig. 8 C: PBS OVA, CTB OVA, pC:pG OVA, pI:pC OVA and pA:pU OVA.
(C) in female C57BL/6 mouse, by the intensity and the feature of OVA and various dsRNA motif inductive t cell responses.In order to measure cell response, by allowing organ pass through 70 microns nylon Falcon filter screen (Becton Dickinson, catalog number (Cat.No.) 352350), use erythrocyte splitting damping fluid (Sigma, catalog number (Cat.No.) R7757) splitting erythrocyte then, obtained splenocyte suspension.By with collagenase (Sigma, catalog number (Cat.No.) C9891) digestion lung tissue, carry out Ficoll-Paque (Amersham Pharmacia, catalog number (Cat.No.) 17-1440-02) gradient centrifugation then, from the relevant Lymphoid tissue of lung, separated lymphocyte.Analyze by ELISPOT, measure t cell response in accordance with the following methods: with rat anti-mouse anti-IFN γ of 4 μ g/ml, IL-2 or IL-4 monoclonal antibody (are respectively BD-PharMingen, catalog number (Cat.No.) 554430, catalog number (Cat.No.) 18161D, catalog number (Cat.No.) 554387) 96-hole 45 microns blended cellulose ester flat board (Millipore, catalog number (Cat.No.) MAHA S4510) is wrapped quilt.Under 37 ℃, after the 10%FCS sealing 1 hour that is present in the Sterile Saline, 5 * 10 5The density of cells/well is added splenocyte suspension, is with or without antigen/peptide.In order to stimulate, used terraced metric antigen (OVA).After stimulating 72 hours, with biotinylated rat anti-mouse cell factor antibody (BD-PharMingen), (BioSourceInt., Camarillo CA) develop to described mensuration with insoluble AEC substrate to use streptavidin-HRP then.The automatization imaging system (Navitar/Micromate) that multiparameter-analysis software (Image Pro, Media Cybernetics) is equipped with in utilization is measured described result.In Fig. 8 D, described result is expressed as the IFN-γ of each spleen and mean value ± SEM (n=4/ group) that the IL-4 spot forms colony (SFC) quantity.Described result represents two independent experiments.
Result among Fig. 8 B-D represents that different synthetic RNAs has enhancement for B and t cell response at the prototype proteantigen.In addition, comprise the different motif of specific nucleotide combination, have special influence at T1 and T2 aspect inducing, and the conversion of immunoglobulin (Ig) isotype takes place subsequently.
Embodiment 20. uses selected synthetic RNA motif can promote the inducing of Tc1 cell of MHC I class-restriction of generation IFN-γ.
(A) handle with pA:pU in the BALB/c mouse of (excite and add booster immunization 2 times) at the HIVgp140 antigen that produces with 10 μ g reorganization-engineerings, studied the cross-excitation that stimulates by the dsRNA motif.Reply by embodiment 19 disclosed ELISPOT assay determinations, use the related peptides R10K of the MHC I class-restriction that comes from the V3 structural domain to carry out stimulated in vitro.In contrast, used the gp140 antigen of dosage coupling.In Fig. 9 A, the result is expressed as the IFN-γ of each spleen and the mean value ± SEM of IL-4 SFC quantity (n=4/ group).
(B) in the C57BL/6 mouse of handling (excite and add booster immunization 2 times) with the complete OV of 100 μ g with pA:pU, analyze by embodiment 19 disclosed ELISPOT, utilize the peptide SUNFEKL[SEQ.I.D.No.2 of MHC I class-restriction] carry out stimulated in vitro, studied the cross-excitation that stimulates by the dsRNA motif.In contrast, used OVA antigen or the aseptic PBS that is present in the salt solution.In Fig. 9 B, the result is expressed as the IFN-γ of each spleen and the mean value ± SEM of IL-4 SFC quantity (n=4/ group).
Result among Fig. 9 A-B represents that selected synthetic RNA motif can promote the enhanced T cellular immunization at the peptide of the different MHC I classes-restriction that is comprised in the big antigen (polypeptide).This immunne response comprises the Tc1 composition, and it is made up of the T cell of the MHC I class-restriction that can produce IFN-γ.
Embodiment 21 represents that different synthetic RNA motifs is unexpectedly in conjunction with different cell receptors; In other words, there is the multiple acceptor that can differentiate the RNA motif.
By facs analysis, by the pA:pU mensuration CD11b of fluorescence-mark +The external combination of APC.Under 4 ℃, ([pA:pU]-F) incubation 30 minutes washs the pA:pU that the isolating APC of MACS-is crossed with 10 μ g/ml marks, and analyzes.In addition, with APC respectively with the pA:pU of the unmarked mistake of 20 or 100 μ g/ml, pA or pI:pC preincubation 10 minutes together, then, the pA:pU dyeing of crossing with mark, and carry out facs analysis.In each picture, represent painted (hollow area) respectively, the curve of the per-cent of undyed (solid area) cell and strong painted APC, X-axis is taken the logarithm.Described result represents independently mensuration twice, and each sample has obtained 10,000 incidents.
Material:
Mouse CD11b, CD11c magneticseparation pearl: be respectively Miltenyi Biotec, catalog number (Cat.No.) 130-049-601, catalog number (Cat.No.) 130-052-001;
ULYSIS nucleic acid marking test kit: Alexa 488, Molecular Probes catalog number (Cat.No.) U21650;
The RNA motif:
PA:pU, (Sigma, lot number 22K4068);
PI:pC, (Sigma, lot number 52K4047);
PA, (Sigma, lot number 22K4022);
FACS damping fluid: PBS, 1%FCS, 0.1% sodiumazide;
MACs damping fluid: PBS, 2mM EDTA, 0.5%BSA;
The collagenase damping fluid: 0.225mg BSA, the 0.0062mg collagenase is present among the 50ml RPMI; With,
70 μ m cell filters: (Falcon/Becton Dickinson, catalog number (Cat.No.) 352350.
Method:
The mark of IRNA motif:
1. in following method, with every kind of RNA motif of ULYSIS Alexa 488 marker marks.
The preparation of II splenocyte:
1. separating Morr. cell and pneumonocyte from 4 female C57 BL/6 mouse;
Different with splenocyte, must shred pneumonocyte, and under 37 ℃, incubation is 30 minutes in the collagenase damping fluid, carries out following steps then;
By 70 μ m falcon cell filters;
Wash and be suspended in again in the MACS damping fluid:
2. according to the method for recommending, carry out mark with CD11b or CD11c specificity MACS pearl;
3. then, with following mass treatment cell:
The pA of unmarked mistake, pA:pU, or pI:pC (20 or 100 μ g/ml), at room temperature mark is 10 minutes;
PA that crosses with 1.5 μ g/ pipe and 10 μ g/ pipe ULYSIS mark or the consumption of pA:pU add respectively, so as with the dyestuff of every kind of motif: the dsRNA ratio is mated.
Mix, and incubation on ice 30 minutes.
Wash once, and be suspended in again in the FACS damping fluid.
The III flow cytometry:
Carry out flow cytometry, so that striving unexpectedly of mensuration/comparison mark RNA motif that cross and unmarked mistake suppresses and the cell receptor combination.
Result among Figure 10 represents that pA:pU and pI:pC are in conjunction with different cell receptors.Because pI:pC, confirmed already that other acceptors different with TLR3 had participated in RNA identification immunologic function in conjunction with TLR3.
The selected synthetic RNA motif of embodiment 22 expressions can be induced the expression in vivo to immunocompetence important chemokine gene.
The RNA that use was extracted from lung tissue after using by respiratory tract in 1 day measures the part rise that the dsRNA motif is expressed chemokine gene by the DNA array technique.(Qiagen, Valencia CA) separate total RNA from lung to utilize the RNeasy test kit.(CA) processing is further purified described RNAs for Stratagene, San Diego by the DNase I with no RNase.(Bethesda, Nonrad-GEArray test kit MD) carries out the DNA array available from SuperArray Inc. by using.Say simply, utilize the synthetic cDNA probe of MMLV reversed transcriptive enzyme and the dNTP mixture that contains vitamin H-16-dUTP.Under 68 ℃, with GEArray film prehybridization 1-2 hour.Hybridize by the cDNA incubation that described film is crossed with vitamin H-mark.The film of hybridizing is with 2 * SSC-1%SDS washed twice, and with 0.1 * SSC-0.5%SDS washed twice.Described film further with alkaline phosphatase-link coupled streptavidin (BioSource Int., Camarillo, CA) incubation together, and finally developing with the CDP-Star chemical luminous substrate.Image-Pro analytical system (Media Cybernetics, Silver Springs, MD) the measured signal intensity of Gel-Pro software is equipped with in utilization.
Result (Figure 11) is expressed as the multiple of the gene expression dose increase of measuring relatively in untreated mouse lung tissue.Compared by dsRNAs (being respectively pA:pU and the pI:pC of 50 μ g) inductive chemokine expression pattern and the LPS inductive chemokine expression pattern by 1 μ g.Optionally represent with successive and discontinuous profile respectively in conjunction with the chemokine of the acceptor on Th1 and the Th2 cell.
Result among Figure 11 represents that pA:pU and pI:pC can induce the expression of multiple chemokine, and its expression pattern is motif-dependent form, and expresses different with LPS (intracellular toxin) inductive.
The specific synthetic RNA motif of embodiment 23 expressions can be mobilized the cotton dress defence that can control pneumovirus infection.
The dsRNA motif has the different abilities of mobilization at the immune defense of influenza infection.With before the influenza infection lung of sublethal dose and 1 day afterwards, by respiratory tract, with the pI:pC of 50 μ g, pA:pU or 50 μ l brine treatment C 3The H/HeJ mouse.In order to carry out virus attack, by respiratory tract sublethal dose (10 4 groupsKnit culture infective dose 50%-TCID 50) work WSN (A/WSN/H1n1) virus infection with the C57BL/6 of Metofane anesthesia and TLR4-/-C 3The H/HeJ mouse.After infecting the 5th day, put to death described mouse, take out lung, homogenate, and-70 ℃ of preservations down.By with the serial dilutions of sample with admissibility mdck cell incubation 48 hours, use chicken red blood cell (available from AnimalTechnologies) to carry out standard hemagglutination then, measure virus titer.Carry out measuring for three times assessment terminal point titre by interpolation technique, and be expressed as TCID 50/ organ (mean value ± SEM; The n=6/ group; Result's representative C 3H/HeJ TLR-4-/-and competence mouse two independent studies of carrying out).Use the TLR4 competence, the C57BL/6 mouse has obtained similar results.
Therefore, result shown in Figure 12 represents by using selected synthetic RNA motif can realize the control that influenza virus is duplicated.
Selected synthetic RNA motif is used in embodiment 24 expression jointly can destroy tolerance to the high dosage standard antigen.
In the mouse with the IgG injection, the dsRNA motif can prevent high-zone tolerance.At first use the hIgG self (solid symbol) of 200 μ g of tolerance dose or with pI:pC or the pA:pU (hollow symbol) of 100 μ g mouse (C57BL/6) is carried out intravenous injection, the hIgG with the 100 μ g that are emulsifiable in the immunogenicity dosage among the CFA (complete Freund's adjuvant) carries out booster immunization by subcutaneous injection then.By the antibody titers of ELISA (as disclosed in the embodiment 19) mensuration to hIgG, its difference is, after injection first, with different intervals, wraps quilt with the hIgG of 10 μ g/ml.In contrast, comprise, and on curve, represent maximum titre (lines of interruption) with the hIgG mice immunized that is emulsifiable in 100 μ g among the CFA.
Result among Figure 13 is expressed as the mean value ± SEM (n=5/ group) of terminal point titre.With TLR4 defective type (C 3H/HeJ) and LPS response type C3H/SnJ mouse obtained similar results.Therefore, the result among Figure 13 represents that the synthetic RNA motif pI:pC that selectes can prevent the tolerance relevant with using large-scale purification albumen usually greatly with pA:pU.
The selected RNA motif of embodiment 25 expressions can induce the different cytokine of people APC to produce.
After differentiation, with the synthetic RNA (pA:pU, pI:pC or pA) of people THP-1 monocyte and different concns incubation together 24 hours, and the collecting cell supernatant liquor.Measure the concentration of IL-12 and TNF-α by ELISA.In Figure 14, the result is expressed as the pg/ml (concentration) of every kind of cytokine and culture condition.
Result among Figure 14 represents the influence to the person monocytic cell of the synthetic RNA motif selected; In addition, this influence is allogenic, depends on the chemical structure of described motif (Nucleotide composition).Select, rather than all synthetic RNA motifs can induce person monocytic cell's IL-12 to produce, it is a kind of important T1 regulating cell factor.
Material:
THP-1 human monocyte cell line: ATCC, catalog number (Cat.No.) TIB-202;
IL-12 cytokine: people ELISA, IL-12 super quick (US) catalog number (Cat.No.) KHC0123;
TNF α cytokine: people ELISA, TNF α catalog number (Cat.No.) KHC3012;
The RNA motif:
PA:pU, (Sigma, lot number 22K4068);
PI:pC, (Sigma, lot number 52K4047); With
PA, (Sigma, lot number 22K4022).
Method:
1. in containing the substratum of 10%FCS, add after the 10ng/ml PMA, allow the THP-1 cell break up.
2. soft washed cell, and add the substratum (HL-1) that does not contain FCS, add treatment agent (RNA motif and contrast) at adherent THP-1 cell top with the concentration of 3-100 μ g/ml.
3. after the incubation 24 hours, the collecting cell supernatant liquor, and measure IL-12 and TNF α concentration by ELISA.
Two kinds of different synthetic RNA motifs of embodiment 26 expression combine people THP-1 monocyte to have shown with the interactional mode of isoacceptor not.
At room temperature, with the synthetic RNA of THP-1 cell and the unmarked mistake of different amounts incubation together 15 minutes.Then, add the pA:pU that mark is crossed,, and carry out fluorescent quantitation by facs analysis at 4 ℃ of following incubations 30 minutes, washed cell.Result among Figure 15 A-15B is the histogram of representing with the maxicell hypotype (A) that is equivalent to and total cell colony (B) form.On each figure, show the per-cent of painted cell.
Result among Figure 15 A-15B represents the pA:pU of unmarked mistake, rather than the pI:pC of unmarked mistake can compete the pA:pU that mark crosses and combine with people THP-1 is monocytic, all on the level of maxicell hypotype and whole colony.
Material:
1.ULYSIS: nucleic acid fluorescent mark (Molecular Probes, catalog number (Cat.No.) U-21650).
2.RNA motif:
PA:pU, (Sigma, lot number 22K4068);
PI:pC, (Sigma, lot number 52K4047);
3.Detoxi-Gel post: (Pierce, catalog number (Cat.No.) 20344).
Method: the I mark of polyadenylic acid-polyuridylic acid (pA:pU):
After removing intracellular toxin, utilize ULYSIS nucleic acid marking system, with Alexa Fluor 488 fluorochrome label pA:pU with the Detoxi-Gel post.
Say simply:
Use sodium acetate and ethanol sedimentation pA:pU down at-70 ℃;
PA:pU is carried out heat denatured, and use Alexa Fluor 488 reagent marks down at 90 ℃; With
Stop described reaction, and the pA:pU that mark is crossed is carried out ethanol sedimentation.
The II cell is handled:
With 2 * 10 6The density suspension THP-1 cell of cell/ml;
With the above-mentioned suspension (5 * 10 of 50 μ l 4Cell) puts into 12 * 75mm test tube;
With the concentration of 20 or 100 μ g/ml, the pA:pU or the pI:pC of unmarked mistake added in the THP-1 cell, and incubation 15 minutes;
Add the pA:pU that the ULYSIS mark is crossed with the concentration of 100 μ g/ml, incubation on ice 30 minutes.
Wash the THP-1 cell once, and be suspended in the FACS damping fluid, carry out flow cytometry then, so that measure the relative fluorescence difference between the different treatment colony.
How the synthetic RNA of adjuvant prepared and purifying before embodiment 27 was illustrated in and uses, so that the performance maximum efficiency.
Total synthetic RNA material is that the standard method by organic synthesis obtains.Then, described material dissolves in no endotoxic Sterile Saline, by removing the intracellular toxin post, is lower than 0.005EU/ μ g up to the concentration of LPS.The mensuration of LPS is measured by standard Limulus and is carried out.Subsequently, by implementing a series of centrifugation step with the filter membrane of specific cell size, the described material of fractional separation (referring to Figure 16).
Useful level part comprises that size is less than 20 to maximum 100bp synthetic RNA.After purifying, measure described material, and confirm: spectrophotometry (OD260nm) by standard test; Gel electrophoresis; Measure by Limulus that to carry out intracellular toxin quantitative; Biological activity on the THP-1 cell (referring to embodiment 25).
Embodiment 28 unexpectedly represents not at the same level part of selected synthetic RNA compound, has different biologic activity according to size.
With the synthetic RNA (pA:pU is according to the method fractional separation of embodiment 27 described disclosures) of people THP-1 monocyte and the different concns of differentiation incubation together 24 hours, and the collection supernatant liquor.(Camarillo CA), measures the concentration of TNF-α by ELISA to use BioSource International test kit.Described result is expressed as every kind of pg/ml (concentration) under the culture condition in Figure 17.
Figure 17 shows that in the result who illustrates level part than small molecular weight of selected synthetic RNA compound has higher biologic activity aspect people's monokaryon THP-1 cells produce cytokine.
Embodiment 29. is with regard to anti--RNA production of antibodies, and selected synthetic RNA motif unexpectedly has different immune characteristics.
By intraperitoneal and subcutaneous [i.p.+s.c.] approach,, and after 1 week, prepare serum sample with hIgG and the immune BALB/c mouse of synthetic RNA (pI:pC or pA:pU) of 50 μ g+50 μ g.In contrast, used with being present in the mouse that the hIgG in the salt solution injected.Measure at pA:pU pI:pC, anti--hIgG and dsRNA IgG antibody titers of pA and hIgG by ELISA.Say that simply (hIgG of 10 μ g/ml or synthetic RNAs) wraps quilt to the hole with antigen, and seal with SeaBlock (Pierce, Rockford, IL, catalog number (Cat.No.) 37527).At room temperature, with the serial dilutions incubation of serum and bronchial perfusate at least 2 hours.After washing, with with alkaline phosphatase (Sigma, catalog number (Cat.No.) A7434) link coupled anti--mouse IgG antibody develops to described mensuration, add substrate (pNPP then, Sigma, and by use the automatization microtiter plate readout instrument of SoftMax software is housed (Molecular Devices ThermoMax) measures catalog number (Cat.No.) N2765).
Result among Figure 18 is expressed as the mean value ± SEM (n=3/ group) of terminal point titre.Result among Figure 18 represents pI:pC, rather than pA:pU induced at it self antibody response, has the cross reaction composition at other RNA motifs.
Load APC, the generation that only when satisfying other conditions, just can cause Tc1 type MHC I class to be replied in the embodiment 30. usefulness reorganization IgG body.
By subcutaneous route, use the immune BALB/c mouse of reorganization IgG-NP (Kd) (referring to Figure 31 A-31D) (the NP peptide is be protected and the conservative epi-position of A type influenza virus) of synthetic RNA (pA:pU or pI:pC) the blended 50 μ g that select with 50 μ g.In contrast, used not contacted antigenic mouse or only with reorganization IgG mice immunized.In immunization after 3 weeks, measure t cell response in accordance with the following methods by the ELISPOT analysis: under 4 ℃, with ELISPOT flat board (Millipore, Molsheim, France) (for anti--IL4,4 μ g/ml are for anti--IFN γ with the anti-cytokine Abs that is present in the purifying among the aseptic PBS (50 μ l/ hole), 8 μ g/ml are available from BD Pharmingen) be incubated overnight together.Next day, with dull and stereotyped 2 times of DMEM substratum washing, and, under 37 ℃, sealed 1 hour with the complete DMEM that contains FBS in 200 μ l/ holes.Prepare single-cell suspension liquid with spleen, splitting erythrocyte, washed cell, counting, and with 5 * 10 5The density in/hole with NP 147-155 peptide or only with the substratum incubation so that the assessment background.Under 37 ℃, 5%CO 2Dull and stereotyped 72 hours of middle incubation.After 3 days, with dull and stereotyped 5 times of PBS-tween20 0.05% (lavation buffer solution) washing, and with the consumption in 100 μ l/ holes, the biotinylated anti-cytokine Abs of 2 μ g/ml in being present in PBS-tween200.05%-FBS 0.1% (ELISPOT damping fluid) is incubated overnight under 4 ℃.
Next day,, and be used in the ELISPOT damping fluid streptavidin with 1: 1000 dilution proportion-HRP incubation 1 hour with dull and stereotyped 5 times of lavation buffer solution washing.(Sigma, St.Luis MO) develop to described reaction, and described dull and stereotyped twice by washing with tap water, termination reaction with 3-amino-9-ethyl carbazole substrate.Then, allow at room temperature dry 24 hours of described flat board.
Data are to use and ImagePro-Plus software are housed ((Navitar, Rochester NY) obtain automation system MD) for Media Cybernetics, SilverSpring.Measure the T cell of generation cytokine and the frequency of NP peptide (the NP peptide is protectiveness and the conservative epi-position of A type influenza virus) reaction, and show at the scale of the peptide that is used to stimulate.Result among Figure 19 is expressed as three multiple mean value ± SEM (n=3 mouse/group).
Use the reorganization IgG of epi-position, caused the generation of Tc2 immunity, but not producing Tc1 replys, this means to have specificity interior formation of body of the I class-peptide complex of stimulation characteristic altogether with NP MHC I class-restriction.After the epi-position (dsRNA1 is that pA:pU and dsRNA2 are pI:pC) that result among Figure 19 is illustrated in the MHC I class-restriction of IgG mediation was sent and passed, the common synthetic RNAs that selectes that uses had promoted effectively inducing of IL-2 and IFN-γ.
Embodiment 31: the APC that is undertaken by Fc γ R loads and by the activation that the RNA acceptor carries out, realize the effective formation of MHC I class peptide and the guidance of resulting t cell response by handling simultaneously.
Never separate spleen APC in the contacted antigenic BALBc mouse body, and under the condition that is with or without the selected synthetic dsRNA (pA:pU) of 50 μ g/ml with 1 μ g NP peptide, or the 50 μ g IgG-NP (Kd) that recombinate spend the night in stripped pulse.Wash described cell, and, use 5 * 10 of equivalent for not contacted antigenic BALB/c mouse by subcutaneous and peritoneal injection 6Cell.After 3 weeks, in accordance with the following methods, reply by the ELISPOT assay determination is described: under 4 ℃, (Millipore, Molsheim France) (resist-IL4 with the anti-cytokine Abs that is present in the purifying among the aseptic PBS (50 μ l/ hole) with the ELISPOT flat board, 4 μ g/ml, anti--IFN γ, 8 μ g/ml are available from BD Pharmingen) be incubated overnight together.Next day,, and under 37 ℃, use the complete DMEM sealing that contains FBS with the consumption in 200 μ l/ holes with dull and stereotyped 2 times of DMEM substratum washing.Prepare single-cell suspension liquid with spleen, splitting erythrocyte, washed cell, counting, and with 5 * 10 5The consumption in/hole and 30 μ g/ml, 10 μ g/ml, or 3 μ g/ml NP peptides or only with the substratum incubation are so that the assessment background level.Under 37 ℃, at 5%CO 2Dull and stereotyped 72 hours of middle incubation.After 3 days, with dull and stereotyped 5 times of PBS-tween20 0.05% (lavation buffer solution) washing, under 4 ℃, with the consumption in 100 μ l/ holes, be that the biotinylated anti-cytokine Abs of 2 μ g/ml is incubated overnight with being present in concentration among the PBS-tween20 0.05%-FBS 0.1% (ELISPOT damping fluid).Next day,, and be used in the ELISPOT damping fluid streptavidin with 1: 1000 dilution proportion-HRP incubation 1 hour with dull and stereotyped 5 times of lavation buffer solution washing.(Sigma, St.Luis MO) develop to described reaction, and described dull and stereotyped twice by washing with tap water, termination reaction with 3-amino-9-ethyl carbazole substrate.Then, allow at room temperature dry 24 hours of described flat board.
Data are to use and ImagePro-Plus software are housed ((Navitar, Rochester NY) obtain automation system MD) for Media Cybernetics, SilverSpring.Result among Figure 20 is expressed as the frequency (mean value ± SEM, n=3 mouse/group) that cytokine produces the spot formation colony of the peptide concentration that stimulates at being used for exsomatizing.In addition, for IFN-γ and IL-4 (arbitrary unit), the average area/colony and the concentration of the peptide that is used to stimulate are mapped.
Result among Figure 20 shows, compares with using peptide itself, and is to APC loadings of exsomatizing, more effective aspect replying at formation MHC I class-peptide complex and generation Tc by reorganization IgG.In addition, the epi-position of being undertaken by IgG/Fc γ R send pass after only MHC I class-peptide complex form, having caused producing IL-4 is not the formation of the Tc2 cell of IFN-γ.Handle APC simultaneously with selected synthetic RNA, caused T cell spectrum to widen the Tc1 cell that produces IFN-γ.
Embodiment 32 expressions excite jointly with IgG-peptide and selected common stimulation motif, have caused the more effective secondary amplification of the T cell of MHC I class-restriction after virus infection.
With reorganization IgG-NP (Kd), pA:pU difference or combination injection BALB/c mouse (50 μ g/ injection).In contrast, used not contacted antigenic mouse.After handling for 3 weeks, with 10 4TCID 50The A/WSN/32H1N1 influenza virus, by the described mouse of respiratory tract infection.Infected 4 days afterwards, by the T cell characteristic in the ELISPOT assay determination spleen, then in accordance with the following methods with the stimulation of exsomatizing of NP peptide: under 4 ℃, (Millipore, Molsheim France) (resist-IL4 with the anti-cytokine Abs that is present in the purifying among the aseptic PBS (50 μ l/ hole) with the ELISPOT flat board, 4 μ g/ml, anti--IFN γ, 8 μ g/ml are available from BD Pharmingen) be incubated overnight together.Next day, with dull and stereotyped 2 times of DMEM substratum washing, and under 37 ℃, with the consumption in 200 μ l/ holes, with the complete DMEM sealing that contains FBS.Prepare single-cell suspension liquid with spleen, splitting erythrocyte, washed cell, counting, and with 5 * 10 5The consumption in/hole with 20 μ g/ml NP peptides or only with the substratum incubation so that the assessment background level.Under 37 ℃, at 5%CO 2Dull and stereotyped 72 hours of middle incubation.After 3 days, with dull and stereotyped 5 times of PBS-tween20 0.05% (lavation buffer solution) washing, under 4 ℃, with the consumption in 100 μ l/ holes, be that the biotinylated anti-cytokine Abs of 2 μ g/ml is incubated overnight with being present in concentration among the PBS-tween20 0.05%-FBS 0.1% (ELISPOT damping fluid).Next day,, and be used in the ELISPOT damping fluid streptavidin with 1: 1000 dilution proportion-HRP incubation 1 hour with dull and stereotyped 5 times of lavation buffer solution washing.(Sigma, St.Luis MO) develop to described reaction, and described dull and stereotyped twice by washing with tap water, termination reaction with 3-amino-9-ethyl carbazole substrate.Then, allow at room temperature dry 24 hours of described flat board.
Data are to use and ImagePro-Plus software are housed ((Navitar, Rochester NY) obtain automation system MD) for Media Cybernetics, SilverSpring.Result among Figure 22 is expressed as the frequency (mean value ± SEM, n=4 mouse/group) of the colony of the T cell formation cytokine that produces NP-specificity MHC I class-restriction.
Result among Figure 21 shows, when using selected synthetic RNA jointly, the sending of the epi-position of the I class restriction of IgG mediation passed at the Tc1 that excites the restriction of I class the most effective aspect replying.After influenza infection, the described precursor that is stimulated increases rapidly.
Selected RNA motif and slotting IgG master's intrachain peptide epitopes are used in embodiment 33 expressions jointly, can the most effectively excite the cytotoxic lymphocyte of the epi-position of identification MHC I class-restriction.
The method that discloses according to the embodiment of front is with reorganization IgG-NP (Kd) immunity and invasion and attack BALBc mouse, and execution in 4 days influenza infection after.The preparation splenocyte, the density of 500 ten thousand/ml is suspended in the HL-1 substratum, and, under the condition that has the 5U/ml recombinant il-2, with the common incubation of NP 147-155 peptide of 10 μ g/ml 5 days.Merging is from the splenocyte of 4 mouse/groups, and in flask incubation.
After amplification, by the cell that the Ficoll gradient centrifugation reclaim to be lived, washing, and under the condition that is with or without NP peptide (20 μ g/ml), with various quantity with the sp20 target cell of fixed qty incubation 5 hours on the V base plate.Flat board is carried out centrifugal after, results supernatant liquor, and by using Promega test kit (catalog number (Cat.No.) G1780) to measure the concentration of LDH.The result is expressed as at different E: the percentage specificity cracking under the T ratio (effector and target cell ratio).
Figure 22 represents to resist-effectively the exciting of virocyte toxicity T cell, need carry out loading in effective body of APC with the epi-position of sending the I class restriction of passing by IgG, and by selected synthetic RNA motif, promptly pA:pU carries out suitable guidance.
Embodiment 34 expression apparatus have the IgG of epi-position of viral MHC I class-restriction and selected synthetic RNA motif inoculation, provide with the infectious provide protection of attacking of prototype virus.
By subcutaneous injection, with the selected immune BALB/c mouse of synthetic RNA (pA:pU) of the reorganization IgG-NP (Kd) of 50 μ g and 50 μ g.3 weeks after the immunity are with 10 4TCID 50Standard infectivity WSN influenza virus invasion and attack mouse, and execution 5 days after.Measure by MDCK hemagglutination, in accordance with the following methods titration Pneumovirinae in the lung homogenate thing: first day, with 2 * 10 4The density of/hole/200 μ l with the mdck cell bed board to 96 hole flat boards, and under 37 ℃, at 5%CO 2Middle incubation 24 hours.With the 10 times diluents of lung homogenate thing in the DMEM substratum of 25 μ l, be placed on incubation (1 minute) on the MDCK flat board of trypsinized in short-term next day, repeat 3 times, and at 37 ℃ of following incubations.After 1 hour, add the DMEM perfect medium of 175l, and under 37 ℃, at 5%CO 2Dull and stereotyped 48 hours of middle incubation.Two days later, adopt chicken red blood cell to carry out hemagglutination and suppress, described chicken red blood cell is at room temperature with from the cell culture supernatant liquid incubation of MDCK flat board 30 minutes, and the result is expressed as the mean value ± SEM (n=4 mouse/group) of lung's virus sum.In contrast, use non-immune mouse.
The result of Figure 23 shows, apparatus has the reorganization IgG of epi-position of viral I class restriction and selected synthetic RNA motif immunity together, caused can limiting virus duplicates after the infectivity invasion and attack the exciting of immunne response.
Embodiment 35. Figure 24 represent to be used to measure the tumor model of rendeing a service based on the molecule of Ig-peptide.
Already with Balb-c mouse (K dRestriction) be used to set up tumor model.Tumour cell (100 ten thousand-1,000 5 hundred ten thousand, be present among the 100 μ L) is injected at flank (referring to the arrow in the photo of top) usually.At first, detect primary tumo(u)r (that is), undertaken quantitatively by measuring tumor size then with slide calliper rule in the injection site by the palpation diseased region.In a series of experiment, with mouse myeloma cell line (SP2/0), the cell of the energy expressing heterologous albumen that cell that untransfected is crossed or stable transfection are crossed (expressing the reorganization IgG of different epitope peptides or complete NP albumen in the CDR3 district of heavy chain) is used for induced tumor in described mouse body.The expression of heterologous protein in the SP2/0 cell provides the relevant antigen (TAA) of specific tumour that is used for measuring various antitumor strategies in immune competence mouse body.Usually, untreated mouse has produced palpable primary solid tumor after 1 week of injection, and it has caused morbid state and dead at 4 time-of-weeks subsequently.Metastasis (referring to Figure 24) has been found in the physical examination that the mouse of injecting carries out.Cultivation is from the Sp2/0 cell of primary tumo(u)r tissue, and the spleen (data are not delivered) that takes out in the mouse body that carries tumour.With reorganization IgG-expression plasmid stable transfection SP2/0 cell, except the special list bit sequence in the CDR3 district that imports heavy chain, for example, outside the NP epi-position (amino acid/11 47-155) that MHCI limits, described plasmid all is identical.The SP2/0 cell can also be with the plasmid transfection stably of the proteic sequence of complete NP that contains the coding WSN virus that is subjected to CMV promotor control.The clone of all transfections has all produced primary tumo(u)r on the framework identical with wild-type SP2/0 cell.
This tumor model is extended to and comprises gland cell system (4T1, ATCC CRL-2539, K dRestriction), confirmed already in the past that it can induce metastatic tumor in the Balb-c mouse.Described 4T-1 clone and SP/0 mentioned above are similar.100 ten thousand-1,000 5 hundred ten thousand 4T-1 injection cells to Balb-c mouse flank, in the time frame that is similar to injection SP2/0 cell, have been produced palpable primary tumo(u)r, and finally caused death.After death the tissue of collecting from various organs shows, can be from spleen, reclaim the 4T-1 (not shown) in lung and the primary tumo(u)r.According to the method described above, use NP-expression plasmid transfection 4T-1 cell stably.The same with the SP2/0 cell, the transfection of 4T-1 cell can not influence the lethality of growth of tumor process and disease.
Embodiment 36 has confirmed after clinical diagnosis by using relevant t cell epitope and the selected common stimulation RNA motif of tumour in the reorganization IgG, to the successful control and the treatment of tumour.
SP2/0 cell (1.5 thousand ten thousand, be present in the 100 μ L volumes) the injection Balb/c mouse that in the CDR3 district of heavy chain (IgNP), has the reorganization IgG of MHC I (Kd) NP epitope peptide with the energy stably express.After injection 7 days, all mouse all have palpable tumour, and described mouse is divided into 3 groups at random: stimulate motif (that is the dsRNA that is made up of poly-pApU) self altogether; The IgTAA albumen (IgNP) of purifying; The two.Treatment time is represented with arrow, and per injection comprises the compound that 50 μ g indicate.The mouse that transfer disease and death take place is represented with " D " in described accompanying drawing.
Being combined in all mouse bodies that have primary tumor when treatment begins of described data representation dsRNA (stimulating motif altogether) and IgTAA (IgNP) all produced the significant protection reaction.Although the mouse with any one independent compound treatment all can fall ill, the mouse of two kinds of compound treatment of usefulness of 100% still lives after handling 3 weeks of beginning, and when in order to measure t cell response they being put to death, they are in good clinical condition.Above result shows, with loading APC (realizing by the Fc acceptor picked-up IgNP by APC) in the TAA body, is not enough to produce Anti-tumor effectively and replys.By tumor rejection and the survival of adopting the mouse performance of handling with pA:pU dsRNA combined I gNP, given prominence to the vital role of common stimulation in performance aspect tumor associated antigen treatment tumour.
In a word, the result among Figure 25 shows, the antigen relevant with tumour carries out loading in the effective body to APC, activates by selected synthetic RNA motif simultaneously, be effectively to control tumor growth and induced tumor to repel necessaryly, and be enough.
Embodiment 37. present embodiments show the suboptimum of tumour antigen are replied under the Asia of tumour cell causes death the inoculation situation, can be by with IgG master's intrachain peptide epitopes with stimulate motif to proofread and correct altogether.
MHC I (the K that in the CDR3 of heavy chain, comprises the WSN virus nucleoprotein with the energy stably express d) the SP2/0 injection cell Balb/c mouse of reorganization IgG (IgNP) of epi-position (amino acid/11 47-155).Described cell inoculation thing is every mouse 100 ten thousand cells (being present in the 100 μ L volumes).Observe described mouse, up to detect palpable tumour in the injection site.At this moment, measure described tumour, and 8 mouse wherein do not handled, and all the other 6 IgTAA that use purifying weekly (that is, and the IgNP of purifying, 2mg/kg) and dsRNA (pApU 4mg/kg) carries out injection in the tumour.Weekly described tumour is measured.
The picture A of Figure 26 is illustrated in 8 mouse, and the 6 inductive tumour progressions and final dead of merely hitting are arranged, and 2 in these mouse have fully spontaneously been repelled described tumour.The picture B of Figure 41 represents weekly 3 times with IgNP/dsRNA (representing by arrow) treatment, 4 moderate stimulations in 6 mouse complete tumor rejection, and in another, produced obviously and disappeared.
Show that at the picture A of Figure 26 and the result among the B antigen that tumour is relevant carries out loading in the effective body to APC, and activate together, can induce efficient immune at tumor associated antigen by selected synthetic RNA.
Embodiment 38 shows that synthesizing RNA with epi-position in the IgG main chain and common pungency treats the mouse of carrying tumour together, has caused the recovery of the state of activation of tumor infiltrating lymphocyte.
Express NP-K with 1,000 ten thousand dThe sp20 transfectoma of epi-position is injected 2 BALB/c mouse.After forming tumour, with selected dsRNA motif (pApU) and 50 μ g " the IgNP "-reorganization IgG-NP (K of 50 μ g that is present in the salt solution d) mouse is carried out injecting in the tumour.Put to death described mouse after 24 hours, tumor resection is used collagenase digesting, by the membrane filtration of 70 μ m, and separates viable cell on the Ficoll gradient.Use anti-TCR, the mAbs of CD25 or isotype contrast pair cell dye, and assess by facs analysis.The result represents with the column diagram form, and has indicated the per-cent of painted cell.
Material:
SP20 clone (ATCC);
2 BALB/c mouse (Harland Sprague Dawley);
70 microns filter membranes of Falcon (Becton Dickinson, catalog number (Cat.No.) 352350);
Collagenase (Sigma, catalog number (Cat.No.) C-9891);
BSA, level part V (Sigma, catalog number (Cat.No.) A-4503);
The collagenase damping fluid: 0.225gm BSA+0.00625gm is present among the 50ml RPMI;
Ficoll-hypaque (1.077, Amersham, catalog number (Cat.No.) 17-1440-02);
The FACS damping fluid: 1% foetal calf serum+0.1% trinitride is present among the PBS;
Antibody: all available from BD Pharmingen; With,
Flow cytometry: FACSCalibur (Becton Dickinson).
Method: tumour cell separates and facs analysis:
In advance 6 weeks carried out tumor inducing according to the method described above;
From BALB/c mouse, separate tumour;
Shred tumour with sterile scissors, and add 10ml collagenase damping fluid;
Under 37 ℃, incubation 40 minutes;
Force tumour to enter in the 50ml test tube with the syringe plunger of 3ml, wash with RPMI simultaneously by 70 μ m Falcon filter membranes;
Wash 1 time, and be suspended in again in the hot RPMI damping fluid of 4ml;
Isopyknic cell suspending liquid is laid on the Ficoll, at room temperature, with the speed of 2000RPM centrifugal 15 minutes;
Separating layer, and with HL-1 damping fluid washing 1 time, and with 2 * 10 6The density of/ml is suspended in the FACS damping fluid again, and carries out flow cytometry;
Remaining cell is used for ELISPOT to be analyzed;
Cell is put into 12 * 75mm test tube with the consumption of 50 μ l/ pipes, and dye with anti--mouse antibodies that the FITC mark is crossed, 2 μ g/ pipes add 1 μ l/ pipe mice serum:
The isotype contrast;
Anti-CD 40;
Anti--CD8;
Anti--CD4;
Anti--CD25;
Anti--TCR γ δ;
Anti--TCR β;
Incubation on ice 30 minutes; With,
With FACS damping fluid washing 1 time, and be suspended in again in the 300 μ l FACS damping fluids.
Result among Figure 27 represents to have the tumor infiltrating lymphocyte of TXi Baoshouti mark TCR β, when handling together with recombination immunoglobulin with tumour associated epitope and selected synthetic dsRNA motif, has obtained the expression of activation tagging CD25.
Embodiment 39 shows that successfully to treat the mouse of carrying tumour with peptide epitopes in the IgG main chain and selected costimulatory molecules relevant with the specific differentiation model of the Tc that also comprises Tc1 except Tc2.
The mouse of tumour can be successfully repelled in execution after the reorganization Ig that has the tumour associated epitope as embodiment 37 described usefulness handles together with selected synthetic dsRNA motif, and by the t cell response of ELISPOT assay determination at the tumour associated epitope.Under 4 ℃, with ELISPOT flat board (Millipore, Molsheim, France) (resist-IL2 and anti--IL4,4 μ g/ml resist-IFN γ with the anti-cytokine Abs that is present in the purifying among the aseptic PBS (50 μ l/ hole), 8 μ g/ml are available from BD Pharmingen) be incubated overnight together.Next day, with dull and stereotyped 2 times of DMEM substratum washing, and under 37 ℃, seal with the complete DMEM that contains FBS in 200 μ l/ holes.
Prepare single-cell suspension liquid with spleen, splitting erythrocyte, washed cell, counting, and with 5 * 10 5The consumption in/hole is with the NP peptide incubation of various concentration.Under 37 ℃, at 5%CO 2Dull and stereotyped 72 hours of middle incubation.After 3 days, with dull and stereotyped 5 times of PBS-tween20 0.05% (lavation buffer solution) washing, and under 4 ℃, with the consumption in 100 μ l/ holes, be that the biotinylated anti-cytokine Abs of 2 β g/ml is incubated overnight with being present in concentration among the PBS-tween20 0.05%-FBS 0.1% (ELISPOT damping fluid).Next day,, and be used in the ELISPOT damping fluid streptavidin with 1: 1000 dilution proportion-HRP incubation 1 hour with dull and stereotyped 5 times of lavation buffer solution washing.(Sigma, St.Luis MO) develop to described reaction, and described dull and stereotyped twice by washing with tap water, termination reaction with 3-amino-9-ethyl carbazole substrate.Then, allow at room temperature dry 24 hours of described flat board.
Data are to use and ImagePro-Plus software are housed ((Navitar, Rochester NY) obtain automation system MD) for Media Cybernetics, SilverSpring.The result is expressed as and is equivalent to IL-4, and the quantity of the spot formation colony of IL-2 and IFN-γ (mean value ± SEM).In contrast, we have used untreated mouse, and they can not repel tumour (n=4/ group).
Result among Figure 28 shows, can successfully repel the Tc1 that mouse that tumor treatment crosses produced at the tumour associated epitope on the therapeutic Ig and reply, and the Tc2 immunity.On the contrary, the mouse that can not repel tumour has only produced the Tc2 immunity.
Embodiment 40 shows that effectively memory response induces after treating the mouse of carrying tumour together with t cell epitope in the IgG main chain and selected common stimulation motif.
According to the disclosed method of embodiment 37, have the reorganization Ig of TAA and selected synthetic RNA motif treatment by injection and carry and express NP-K dThe sp2/0 mouse of the tumour of TAA.After tumor rejection, reach NP-Kd by the offside land for building firing table of making a bet Table1,000 5 hundred ten thousand described mouse of SP2/0 cell invasion of position.Simultaneously, the not contacted antigenic mouse of 4 contrasts is injected the cell of the same type of tumorigenesis/lethal dose equally.Monitor the development and the size of described tumour, and be expressed as diameter (mm) and the time that begins from stimulation.
Result among Figure 29 shows that the treatment of passing through to be indicated successfully induced tumor rejection, can effectively prevent the invasion and attack subsequently of identical tumour then, shows to have produced effective immunological memory.
Embodiment 41 shows and it is shocking, IgG by having TAA and stimulant inductive tumor rejection have altogether caused the tumour variant of multiple shortage TAA or have the cross-protection of the variant of TAA.
To the embodiment 40 described mouse that can avoid the homology invasion and attack, all ages show to lack the identical tumour cell of TAA (lacking antigenic variant) or have shortage NP-K with 1,000 5 hundred dThe tumour cell of the TAA variant of epi-position carries out continued stimulus.In addition, in contrast, with different types of tumors clone (4T-1 gland cancer) invasion and attack mouse, shown in the appended table of Figure 30 A.Under each situation, all comprise not contacted antigenic contrast.
Analyze by ELISPOT, use TAA (NP-Kd peptide), HA (peptide of MHC II class-restriction), or from the splenocyte suspension that the protein extract of cell pyrolysis liquid stimulates has assessed the T cellular immunity of avoiding the mouse that the kinds of tumors variant attacks.Under 4 ℃, with ELISPOT flat board (Millipore, Molsheim, France) (resist-IL2 and anti--IL4,4 μ g/ml resist-IFN γ with the anti-cytokine Abs that is present in the purifying among the aseptic PBS (50 μ l/ hole), 8 μ g/ml are available from BD Pharmingen) be incubated overnight together.Next day, with dull and stereotyped 2 times of DMEM substratum washing, and under 37 ℃, with the consumption in 200 μ l/ holes, with the complete DMEM sealing that contains FBS.
Prepare single-cell suspension liquid with spleen, splitting erythrocyte, washed cell, counting, and with 5 * 10 5The consumption in/hole is with the antigen incubation of various concentration.Under 37 ℃, at 5%CO 2Middle cultivation incubation 72 hours.After 3 days, with dull and stereotyped 5 times of PBS-tween20 0.05% (lavation buffer solution) washing, and under 4 ℃, the consumption in 100 μ l/ holes is that the biotinylated anti-cytokine Abs of 2 β g/ml is incubated overnight with being present in concentration among the PBS-tween20 0.05%-FBS 0.1%6 (ELISPOT damping fluid).Next day,, and be used in the ELISPOT damping fluid streptavidin of 1: 1000 dilution proportion-HRP incubation 1 hour with dull and stereotyped 5 times of lavation buffer solution washing.(Sigma, St.Luis MO) develop to described reaction, and described dull and stereotyped twice by washing with tap water, termination reaction with 3-amino-9-ethyl carbazole substrate.Then, allow at room temperature dry 24 hours of described flat board.
Data are to use and ImagePro-Plus software are housed ((Navitar, Rochester NY) obtain automation system MD) for Media Cybernetics, SilverSpring.The result is expressed as and is equivalent to IL-4, and the quantity of the spot formation colony of IL-2 and IFN-γ (mean value ± SEM).In contrast, used the untreated mouse (n=4/ group) that can not repel tumour.In contrast, comprise not contacted antigenic mouse.The result is with the quantity of cytokine production cell/organ ((the n=3/ group) that the form of mean value ± SEM) is represented.
Result among Figure 30 A-30B (comprising the table among Figure 30 A) shows, the immunity that occurs after the treatment of indicating that causes tumor rejection has caused the avoiding of the invasion and attack of antigenic variant, and relevant with the overall amplification of cytokine production cell.This shows by the method for recommending, and the lymphocytic repertoire of Anti-tumor is widened be not the relevant antigen of tumour that carried by the immunotherapy molecule.

Claims (21)

1. the composition that comprises double-stranded RNA is used for strengthening the experimenter to the purposes in the medicine of antigenic t cell response in preparation.
2. purposes as claimed in claim 1, wherein said medicine further comprises described antigen.
3. purposes as claimed in claim 1, wherein, described antigen is used or is contacted after using double-stranded RNA to described experimenter.
4. purposes as claimed in claim 1, wherein, described double-stranded RNA comprises poly-VITAMIN B4 and poly-uridylic.
5. purposes as claimed in claim 1, wherein, described double-stranded RNA comprises poly-guanine and poly-cytosine(Cyt).
6. purposes as claimed in claim 1, wherein, described medicine can strengthen replys described antigenic Th1.
7. purposes as claimed in claim 1, wherein, described medicine can strengthen replys described antigenic Tc1.
8. non-infectious antigen and the double-stranded RNA composition that comprises poly-VITAMIN B4 and poly-uridylic or polyinosine and poly-cytosine(Cyt) are used for preventing purposes to the pharmaceutical composition of non-infectious antigenic high-zone tolerance in preparation.
9. purposes as claimed in claim 8, wherein, described medicine can prevent T cell anergy.
10. purposes as claimed in claim 8, wherein, described antigen is virus.
11. purposes as claimed in claim 8, wherein, described dsRNA is pA:pU.
12. a composition that is used to strengthen to antigenic t cell response comprises the dsRNA sequence of being made up of poly-VITAMIN B4 and poly-uridylic.
13. as the composition of claim 12, wherein, described composition also comprises antigen.
14. as the composition of claim 12, wherein, described antigen is to use in carrier that can be medicinal.
15. as the composition of claim 12, wherein, described antigen is used in immunoglobulin (Ig).
16. as the composition of claim 12, wherein, described can medicinal carrier be IgG.
17. as the composition of claim 12, wherein, described antigen is the tumour associated epitope.
18. as the composition of claim 13, wherein, described antigen is virus.
19. as the composition of claim 12, wherein, described antigen is the relevant t cell epitope of tumour.
20. one kind is used for comprising the dsRNA sequence at the composition of experimenter's enhancing to antigenic t cell response, it further comprises polyinosine and poly-cytosine(Cyt).
21. as the composition of claim 20, wherein, described composition also comprises antigen.
CNB038109174A 2002-03-15 2003-03-14 Compositions and methods to initiate or enhance antibody and major-histocompatibility class i or class ii-restricted t cell responses by using immunomodulatory, non-coding RNA motifs Expired - Fee Related CN100376594C (en)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Cellular responses to double-stranded RNA. HAINES,D.et al.J.Cell.Biochem.,Vol.46 No.00. 1991 *

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