CN102573902A - Immunoconjugates comprising poxvirus-derived peptides and antibodies against antigen-presenting cells for subunit-based poxvirus vaccines - Google Patents

Abstract

The present invention concerns methods and compositions for subunit-based vaccines for inducing immunity against poxvirus infections, such as smallpox. Preferred embodiments concern immunoconjugates comprising one or more subunit antigenic peptides attached to an antibody or fragment thereof that targets antigen-producing cells (APCs). More preferably, the antibody binds to HLA-DR and the antigenic peptide is from an immunomodulating factor, such as the viral IL-18 binding protein (vIL18BP). However, mixtures of antigenic peptides from different viral proteins may also be used. The vaccine is capable of inducing immunity against poxvirus without risk of disseminated infection in immunocompromised hosts or transmission to susceptible contacts.

Description

Be used for based on the poxvirus vaccine of subunit comprise the poxvirus derived peptide and to the immune conjugate of the antibody of antigen-presenting cell

Related application

The application requires the 12/754th of submission on April 5th, 2010; The 12/754th of No. 140 U.S. Patent applications, submission on April 6th, 2010; The 61/258th of the 61/258th, No. 369 U.S. Provisional Patent Application of submitting in No. 740 U.S. Patent applications and on November 5th, 2009, submission on November 6th, 2009; The priority of the 61/378th, No. 059 U.S. Provisional Patent Application that No. 729 U.S. Provisional Patent Application and on August 30th, 2010 submit to.The text full content of each priority application is incorporated this paper into way of reference.

Background

Invention field

The present invention relates to be used to treat and/or prevent design, generation and the application based on the vaccine of subunit of poxvirus infection (including but not limited to variola).In preferred embodiments, vaccine comprises the immune conjugate from the subunit antigen peptide of one or more virus proteins.In more preferred, virus protein is an immunoregulatory factor, like viral IL-18 conjugated protein (vIL18BP), but can use alternative virus protein, like virus envelope protein.In other alternate embodiment, possibly comprise combination from the antigenic peptides of more than one virus proteins (like immunoregulatory factor and envelope protein) based on the vaccine of subunit.The virus antigen peptide is connected to antibody or its Fab, thereby said subunit targeted is produced cell (APC) to antigen.In most preferred embodiment, based on the vaccine of subunit and have to antigenic antibody of HLA-DR or antibody fragment, like L243 antibody; But one skilled in the art will recognize that other APC targeting antibodies is known, and can use.The use of immune conjugate provides significantly the immunogenicity that increases and to the improved immune system response of virus antigen, avoids contacting the individual possibility of infection of the hypoimmunity of live-virus vaccine simultaneously.Preferably, induce immunity in vivo effectively based on the vaccine of subunit, and prevent to receive it to infect variola and/or other poxvirus.

Correlation technique

One group of complex virus with cross reacting antigen is that vaccinia subgroup virus comprises vaccinia virus (VV), monkey pox virus and causes variola (variola, virus VAR).Variola no longer is a kind of abiogenous infectious disease; It has been lasted till that a large-scale The Immune Programming till 1978 eliminates; The routine vaccination of world population from that time stops (Minor, 2002, British Med J 62:213-224).At that time, remaining virus stock solution used was deposited in the U.S. and the former Soviet Union.The threat of the bioterrorism that occurs recently, the monkeypox (CDC MMWR2003) that breaks out recently; The fatal character of the following day blossom disease of especially only inoculating less than half the population according to estimates in the whole world of situation has reignited the concern of protecting to other dangerous member's of VAR or this virus family vaccine for development.

The antismallpox vaccine representative at present of adopting active VV to variola carry out immunity the most effectively means (Rosenthal etc., 2001, Emerg.Infect.Dis.7:920-926).This vaccine produces of short duration viremia, and it is able to disappear in most of individualities, and stays permanent immunity.Yet this vaccine is also owing to comprising that general viremia and dead serious adverse reaction cause safety problem (He etc., 2007, JID 196:1026-1032; Rosenthal etc., 2001).Therefore, if objective situation requires to carry out the population immunity, just must exploitation substitute vaccination strategies.

The several method that substitutes vaccine was attempted, or just under development.Attenuation poxvirus form with disappearance or mutant gene like Akhara improvement cowpox (MVA) (Grandpre etc., Vaccine 27:1549-56,2009), can be given the partial immunity power to the virulent strain of poxvirus.The immunity of carrying out with inactivation of viruses has obtained research, but it does not give the protection with the live virus same degree; No matter use any ablation method, all need many 10 ³-10 ⁴The inactivation of viruses of individual unit protects mice to avoid exciting influence (Turner etc. 1970, J Hyg.Camb68:197-210).This fact means, carries out the immune protection that is obtained with challenge virus and comprises that nonactive virus can not produce or the non-existent factor in nonactive virus.Poxvirus produces a series of secreted host immune response modifying factors, and it can neutralize the host cell factor and congenital defense mechanism.Through weakening host's the first line of defence, virus possibly can be set up infection (as in mucosa), and the infectivity of beginning phase I is duplicated in host cell.

Existence is for the needs of the vaccine that is directed against poxvirus (like variola), and said vaccine is more effective than inactivation of viruses, but the safety issue that can avoid live-virus vaccine to occur.

Summary of the invention

The invention discloses improved compositions and its method for using to the subunit vaccine of poxvirus (like variola).In preferred embodiments, this vaccine comprises one or more subunit antigen peptides and conjugated antigen is delivery cell (APC), like the antibody of BMDC (DC) or antibody fragment coupling to form immune conjugate.Use vaccine to the experimenter and can induce immunity, and effectively prevent or treat poxvirus infection poxvirus.Randomly, this vaccine maybe and have one or more adjuvants, like aluminium hydroxide, CpG DNA, calcium phosphate or based on the adjuvant (for example, De Shi Lactobacillus bulgaricus (L.delbroeckii/bulgaricus)) of antibacterial.

A kind of host immune regulatory factor that VV and VAR produce is viral interleukin-18 conjugated protein (vIL18BP, a vaccinia virus C12L gene).The same with other virus host defence regulatory factor; This gene is expressed at the commitment that infects, and it weakens host immune power through neutralizing crucial pro-inflammatory cytokine IL-18, and said pro-inflammatory cytokine stimulates NK, CD8 and Th1CD4 cell to produce interferon-(IFN); It and then active antigen are delivery cell (APC) and other cell; And guiding is to immunoreation (Born etc., 2000, the J.Immunol.164:3246-3254 of Th1 type; Scott, 1991, J Immunol147:3149-3155; Pien etc., 2002 .Immunol.169:5827-5837; Xiang and Moss, 1999, Proc.Natl.Acad.Sci.USA 96:11537-11542).In preferred embodiments, select the epi-position of subunit antigen peptide with simulation vIL18BP.Other exemplary host immune regulatory factor and locus_tag identifier thereof provide in table 1.

In other preferred embodiment, the subunit antigen peptide comes from virus immunity and regulates albumen.Those skilled in the art will recognize that various virus immunities adjusting albumen are known and can use.Limiting examples comprises interferon-(IFN-γ) receptor homolog thing (B8R gene), Complement Regulatory Protein congener (B5R gene) and serpin (B13R, B14R, B22R gene).Multiple poxvirus immune modulator has been able to report, but its influence to virus immunity originality is never characterized well.(referring to J Virol79:6554-59 such as for example Jackson, 2005; B12R gene (serine/threonine protein kitase); B15R gene (IL-1 and IL-6 receptor); B16R gene (IL-1 receptor), B18R gene (IFN-α receptor), B19R gene (IL-1 and IL-6 receptor, IFN inhibitive factor).

Table 1. poxvirus immune modulator

In alternate embodiment, the subunit antigen peptide possibly come from virus envelope protein or other virus protein.Limiting examples comprises the protein product of D8L, A27L, L1R and A33R gene.Those skilled in the art will recognize that said various poxvirus genome and protein DNA and aminoacid sequence are well known in the art and can openly obtain (about the complete genome group sequence of vaccinia virus WR and coded protein sequence, referring to for example GenBank accession number AY243312).

The antibody component of immune conjugate guides to APC with complex, and wherein the antigenic peptides component is through processing to cause poxvirus and/or to express the immunoreation of the infected cell of target antigen.Various APC targeting antibodies are known in the art, as combining to be selected from the antigenic antibody by the following group of forming: HLA-DR, CD74, CD209 (DC-SIGN), CD34, CD74, CD205, TLR2 (toll appearance receptor 2), TLR4, TLR7, TLR, BDCA-2, BDCA-3 and BDCA-4.In more preferred, antibodies is selected from the antigen of HLA-DR and CD74.In most preferred embodiment, antibodies HLA-DR.

In some preferred embodiments, poxvirus vaccine comprises humanization, people or inosculating antibody-HLA-DR antibody, like L243 antibody.L243 antibody is described that (for example United States Patent (USP) the 7th, 612, No. 180; Embodiment part is incorporated this paper into way of reference) and be characterised in that have heavy chain complementary determining region (CDR) sequence C DR1 (NYGMN, SEQ ID NO:1), CDR2 (WINTYTREPTYADDFKG; And CDR3 (DITAVVPTGFDY, SEQ ID NO:3) and light chain CDR sequence C DR1 (RASENIYSNLA SEQ ID NO:2); SEQ ID NO:4), CDR2 (AASNLAD, SEQ ID NO:5); And CDR3 (OHFWTTPWA, SEQ ID NO:6).Yet, can use other anti--HLA-DR antibody as known in the art (referring to for example United States Patent (USP) the 6th, 416,958,6,894,149,7,262, No. 278, the embodiment part is incorporated this paper into way of reference respectively).

In other preferred embodiment, poxvirus vaccine comprises humanization, people or inosculating antibody-CD74 antibody, like LL1 antibody.LL1 antibody is described that (for example United States Patent (USP) the 7th, 312, No. 318; Embodiment part is incorporated this paper into way of reference) and be characterised in that have light chain CDR sequence C DR1 (RSSQSLVHRNGNTYLH, SEQ ID NO:7), CDR2 (TVSNRFS; And CDR3 (SQSSHVPPT, SEQ ID NO:9) and heavy chain CDR sequence C DR1 (NYGVN SEQ ID NO:8); SEQ ID NO:10), CDR2 (WINPNTGEPTFDDDFKG, SEQ ID NO:11); And CDR3 (SRGKNEAWFAY, SEQ ID NO:12).Perhaps, can utilize other anti-CD74 antibody or to the antibody of other APC or DC related antigen (referring to for example LifeSpan Biosciences Inc., Seattle, WA; BioLegend, San Diego, CA; Abcam, Cambridge, MA).

In various embodiments, antibody or its Fab possibly be chimeric, humanization or people's antibody or its Fab.Use chimeric antibody to be superior to parent Mus source antibody,, therefore can not cause the same strong human anti-mouse antibody with Mus source antibody (HAMA) reaction because they possess people's antibody constant region sequence.In order further to reduce the probability of bringing out the HAMA reaction, it is preferred using humanized antibody.Discuss as following, to make Mus source antibody humanization's technology be well known in the art through Mus source framework and constant region sequence are replaced with corresponding human antibody framework and constant region sequence, and be applied to numerous Mus source anticancrins.The antibody humanization also possibly relate to and uses the corresponding residue from parent Mus source framework region sequence to replace one or more people's framework amino acid residues.Also like following discussion, the technology that produces people's antibody is also known, and this antibody-like can be incorporated in the theme poxvirus vaccine construct.

Other embodiment relates to the DNA sequence of encoding fusion protein (like antibody-subunit antigen peptide fusion protein), contains the carrier and the host cell of said DNA sequence, and the method for making the fusion rotein be used to produce poxvirus vaccine.In some embodiments, use DNL (butt joint-with-locking) technology to make under the situation of subunit vaccine, fusion rotein can comprise DDD (dimerization and butt joint territory) part or AD (grappling territory) part.In alternate embodiment, can form immune conjugate through the chemical crosslinking of for example antibody or antibody fragment and antigenic peptides.Detailed Description Of The Invention

Definition

Term " " and " said (being somebody's turn to do) " that this paper uses possibly be meant odd number or plural number, only represent odd number only if context spells out in addition.

The term " about " that this paper uses is that exponential quantity adds or deduct 10 (10%).For example, " about 100 " are meant any numeral between 90 and 110.

AntibodyBe meant total length (be spontaneous generation or form through normal immunoglobulin gene fragment regrouping process) immunoglobulin molecules (like IgG antibody), or the immunocompetence of immunoglobulin molecules, antigen-binding portion thereof, like antibody fragment.

Antibody fragmentBe the part of antibody, like F (ab ') ₂, F (ab) ₂, Fab ', Fab, Fv, scFv etc.No matter structure why, and antibody fragment combines the same antigen by complete antibody identification.Therefore, this term uses with the free burial ground for the destitute with " antigen binding antibody fragment ".Term " antibody fragment " also comprises the isolated fragment of being made up of the variable region, the recombinant single chain peptide molecule (" scFv albumen ") that is connected through connection peptides like " Fv " fragment be made up of heavy chain and variable region of light chain and light chain and variable region of heavy chain.The term " antibody fragment " that this paper uses does not comprise that no antigen combines active antibody moiety, like Fc fragment or single amino acids residue.Other antibody fragment, for example the single domain antibody fragment is known in the art, and in the construct that can be used for being advocated.(referring to for example Muyldermans etc., TIBS 26:230-235,2001; Yau etc., J Immunol Methods281:161-75,2003; Maass etc., J Immunol Methods 324:13-25,2007).

Term Antibody fusion proteinCan refer to have the antigen binding molecules that reorganization that identical or different specific one or more identical or different single-chain antibodies or antibody fragment link to each other produces.The valence state of fusion rotein indication fusion rotein is with respect to single antigen or brachium conjunctivum or the quantity in site, i.e. monovalence, bivalence, trivalent or multivalence that epi-position had.The polyvalency of antibody fusion protein is meant that it can utilize the multiplephase mutual effect when conjugated antigen, thereby improves and the bonded affinity of antigen (avidity).Specificity indication antibody fusion protein can bonded antigen or the quantity of epi-position, i.e. monospecific, bispecific, tri-specific, polyspecific.Use these definition, for example the natural antibody of IgG is a bivalence, because it has two brachium conjunctivums, but it is a monospecific, because it combines an epi-position.Monospecific, multivalence fusion rotein have an above binding site for epi-position, but only combine an epi-position.Fusion rotein possibly comprise the multivalence or the polyspecific combination of monospecific antibody component, different antibodies component, or a plurality of copies of same antibody component.In addition, fusion rotein can comprise antibody or antibody fragment and subunit peptide antigen.Yet this term is not restrictive, and multiple protein or peptide effector can be incorporated in the fusion rotein.In another limiting examples, fusion rotein can comprise the AD or the DDD sequence of the DNL construct that is used to produce following discussion.

Chimeric antibody is the variable domain (comprising complementary determining region (CDR)) that contains the antibody (preferred rodent animal antibody) that derives from species, and the constant domain of antibody molecule derives from the recombiant protein of the constant domain of people's antibody.For veterinary applications, the constant domain of chimeric antibody possibly come from other species, like cat or Canis familiaris L..

Humanized antibody is the recombiant protein that shifts the pure man heavy chain and light chain variable territory (for example framework region sequence) from the heavy chain and the light chain variable chain of rodent animal antibody from the CDR of the antibody of species (for example rodent animal antibody).The constant domain of antibody molecule is derived from people's antibody.In certain embodiments, from the desirable substitution people of the framework region amino acid residue antibody framework region sequence of the limited quantity of parent (rodent) antibody.

People's antibody is for example by the antibody that obtains with the transgenic mice that produces human antibodies specific in response to antigen stimulation through " through engineering approaches ".In this technology, the element of people's heavy chain and light chain gene seat is introduced in the mouse species of the targeted disruption that comprises endogenous Mus source heavy chain and light chain gene seat that is derived from embryonic stem cell line.Transgenic mice can synthesize the specific people's antibody that has to specific antigen, and said mice can be used for producing the hybridoma of secretion people antibody.The method that obtains people's antibody from transgenic mice is by Green etc., Nature Genet.7:13 (1994), and Lonberg etc., Nature 368:856 (1994) and Taylor etc., Int.Immun.6:579 (1994) describes.Fully human antibodies also can pass through gene or chromosome transfection method, and display technique of bacteriophage makes up, and said method and technology all are known in the art.About from immunoglobulin variable domain gene external the generations people antibody of spectrum and its fragment from epidemic disease donor rather, referring to for example McCafferty etc., Nature 348:552-553 (1990).In this technology, the antibody variable domain gene is cloned in the main or less important coat protein gene of filobactivirus with frame, and is showed on the surface of phage particle with the functional antibodies pieces.Because filamentous particle contains the single stranded DNA copy of phage genome, so based on the selection of the functional character of antibody also cause the encoding selection of gene of the antibody that represents these character.Like this, some character of phage simulation B cell.Phage display can be carried out in many ways, about comment, and referring to for example Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).People's antibody also can be through producing at external activatory B cell.Referring to United States Patent (USP) No. 5567610 and No. 5229275, the embodiment part is incorporated this paper into way of reference.

The accompanying drawing summary

Fig. 1. be derived from the combination and the absorption of the peptide of vIL18BP sequence (SEQ ID NO:23).(A) vIL18BP110 (SEQ ID NO:16) combines with the T2 cell.Shown peptide (TT830, SEQ ID NO:19; VA4L229, SEQ ID NO:18; VIL18BP008, SEQ IDNO:13; VIL18BP105, SEQ ID NO:15; VIL18BP110, SEQ ID NO:16; VIL18BP117, SEQ ID NO:17) hatch 24h with the T2 cell.Show the relative abundance of HLA-A02 in the T2 cell.Bar diagram is from left to right represented the concentration of from 0 to 40 μ g/mL with the peptide of 10-μ g/mL incremental increase respectively.(B) vIL18BP105 (SEQ IDNO:15) represents the highest donor PBMC absorption.With shown in after the biotinylation peptide is hatched 24h, estimate the parallel double duplicate samples.NJ01, NJ04, NJ07 and NJ08.After adding avidin-FITC conjugate, through the flow cytometry result.The fluorescent value of each peptide equals peptide in the same experiment and handles the fluorescent value that the fluorescent value of cell deducts untreated cell.Peptide concentration is 20 mcg/ml.

Fig. 2. the PBMC from the inoculation donor breeds when hatching with viral peptide.Will be from PBMC and 10 μ g/mL specified polypeptides (vA27L003, the SEQ ID NO:20 of inoculation (A) with the load C FSE that inoculates (B) people donor; VD8L118, SEQ ID NO:22; VIL18BP105, SEQ ID NO:15 or contrast) hatched together 5 days.Harvesting, and utilize flow cytometry (meansigma methods ± SD).Bar diagram shows by following order: packless bar diagram, culture medium contrast; The filled black bar diagram, 2.5mg/mL peptide (or SEA); The light grey bar diagram of level-hacures, the 5.0mg/mL peptide; Vertically-and hacures Dark grey bar diagram, the 10.0mg/mL peptide.(C) from the result of independent experiment, wherein hatch to measure the expression of the cell within a cell factor and activation mark with vD8L118 (SEQ ID NO:22) from the cell of designated samples.The result is shown in (D8L, vD8L118 peptide, SEQ ID NO:22) in the embedded table (C).* than culture medium contrast, cell mean P＜0.05 (t-check).

Fig. 3. reply the phenotype that CD8+IFN-γ+cell has TEM or CD45RA-terminally differentiated cells.Express to CD45RA and CCR7, the CD8+PBMC from the inoculation donor is assessed.Number is represented the percentage ratio of TCS.For TEM colony, vD8L118 is than P＜0.019 (ANOVA) (left lower quadrant) of culture medium contrast.

The CD107a of Fig. 4 .CD8+ cell expresses.Than the potential mark CD107a of threshing, IFN-γ, the IL-2 of assessment CD8+ cell colony.The representative of number and bar diagram value is to cell percentage ratio in the door of following cell: (A) CD8+IFN-γ+cell, (B) CD8+IFN-γ-cell and (C) CD8+IL-2+ cell.* than culture medium contrast, cell mean P＜0.04 (t-check).

Fig. 5. peptide antibody is present in the serum of inoculating donor.To and hatch from serum 1: 200 dilution of not inoculation or inoculation donor: (A) peptide vA27L003 (SEQ ID NO:20), (B) peptide vD8L110 (SEQ ID NO:21) and (C) peptide vIL18BP102 (SEQ ID NO:14) to the peptide on being fixed in the 96-orifice plate among the improvement ELISA of following peptide.Round dot is represented the A450 of each donor.* than inoculator not, P＜0.03 (ANOVA).Do not inoculate donor: 213,704,220; Inoculation donor: 05,12A, 12B, 19,26,720,308,416 and 920.Peptide vD8L110 and vIL18BP105 are the 25-aggressiveness that comprises vD8L118 and vIL18BP105 complete sequence.

Fig. 6. with respect to the HLA-DR04tg splenocyte propagation of vIL18BP105.With the HLA-DR04tg mice of vIL18BP105-L243 conjugate (conjugate) or free vIL18BP105 (dissociating) inoculation, originally HLA-DR04tg mice (HLA-DR04tg originally) and wild type C57BL/6J (WT originally) (n=3 mice/group).Use the splenocyte through the CFSE labelling of hatching to carry out repeated trials three times with the peptide of variable concentrations.The result is representative value (n=3, the meansigma methods ± SD) of 3 independent experiments.

Fig. 7. booster immunization is 7 and 14 days afterwards for the first time, produces with the peptide specific serum antibody in the HLA-DR04tg mice of CIL18BP105 (conjugate) and IL18BP105 (dissociating) immunity.Originally the HLA-DR04tg mice serum is with comparing (originally).Experiment repeats (n=3, meansigma methods ± SD) for three times with the serum that compiles.

Fig. 8. the serum antibody from immune mouse combines with complete vIL18BP is proteic.To identification complete vIL18BP proteic antibody, to independent L243 antibody, vIL18BP105 peptide (SEQ ID NO:15), and the viral IL18BP105 peptide (CIL18BP105) of L243 antibody coupling, single culture base mice immunized or originally the serum of mice test.

Fig. 9. the immune conjugate based on liposome of subunit vaccine.(A) peptide-L243 antibody coupling matter of liposome displaying.(B) the naked peptide that does not have antibody of liposome displaying.

Poxvirus vaccine

The subunit antigen peptide

Poxvirus produces a series of secreted host immune response modifying factors, and it can neutralize the host cell factor and congenital defense mechanism.The first line of defence that weakens the host can make virus set up to infect (as in mucosa), and then begins the infectivity of phase I and duplicate.VV is viral interleukin-18 conjugated protein (vIL18BP, a vaccinia virus C12L gene) with a kind of factor that other poxvirus produces, and it expresses J Immunol164:3246-54 such as (, 2000) Born at the commitment that infects.It works through neutralizing crucial pro-inflammatory cytokine IL-18; Said pro-inflammatory cytokine stimulates NK, CD8 and Th1CD4 cell to produce interferon-(IFN-γ); Its guiding is to acquired immunity (Livingston etc., J Immunol168:5499-5506,2002 of Th1 type; Pien etc., J Immunol 160:5827-37,2002; Scott, JImmunol 147:3149-55,1991; Turner etc., J Hyg Camb 68:197-210,1970; Xiang and Moss, PNAS USA 96:11537-542,1999).

Whether the host response of researching and solving vIL18BP described in the embodiment below involves the problem for the resistance of poxvirus infection.If like this, substitute the vaccine strategy and should comprise this factor and/or similar antigen.Recently to be that I type IFN-is conjugated protein be absolutely necessary (Xu etc., JExp Med 205:981-92,2008) and can be the material standed for that is contained in the subunit poxvirus vaccine for pathogenicity the another kind of vaccinia subgroup virus host defense regulatory factor of report.Discuss as following, other known virus protein also can be used as based on the material standed for of the subunit antigen peptide of the poxvirus vaccine of subunit and in being included in.

Described in following examples, whether can cause that through research vIL18BP antigenic peptides anamnesis reaction through inoculation people experimenter's PMBC (PBMC) and serum comes the vaccine method based on the subunit peptide of test person immunity.Cell-mediated immunity is to the importance in the resistance of poxvirus still under study for action, but immunity needs antibody response to obtain necessarily generally acknowledging (Chaudhri etc., J Virol 80:6339-44,2006; Combadiere etc., J Exp Med 199,1585-89,2004; Kim etc., Clin Vaccine Immunol 13:1172-74,2006).

Except the vIL18BP-derived peptide, the peptide that derives from other VV gene is that D8L and A27L also are able to test.D8L albumen is for virus absorption and to get into cell be important, and has shown and can in the mouse model of poxvirus infection, cause intensive protective immunity (Kan-Mitchell etc., J Immunol 172:5249-61,2010; Berhanu etc., J Virol82:3517-29,2008).A27L albumen also is important for virus absorption and assembling, and to its antibody protective immunity (Berhanu etc., J Virol 82:3517-29,2008 is provided; Chun g etc., J Virol 72:1577-85,1998; Scott, J Immunol 147:3149-55,1991).

Overall goal of the present invention is to select and develop to supply to be contained in T-cell (HLA-combination) and the B-cellular antigens peptide in the polyepitope vaccines specification.Peptide has the advantage that is easy to synthesize, modify and be combined into the former complex of multi-resistance relatively.In order to improve its immunogenicity, peptide is connected as being directed against the antigenic antibody of HLA-DR with the antibody of targeting APC.

The antibody of targeting APC

As special antigen-presenting cell, BMDC (DC) is being brought into play very important effect in starting innate immunity and adaptive immunity, and has been used to produce effective vaccine (Vulink etc., Adv Cancer Res.2008,99:363-407; O ' Neill etc., Mol Biotechnol.2007,36:131-41).In vivo the antigen targeted is represented a kind of promising inoculation method to APC and DC; The stripped antigen load and the cultivation of costliness because it can avoid requiring great effort; And help immunotherapy large-scale application (Tacken etc., Nat Rev Immunol.2007,7:790-802).The more important thing is that the inoculation of APC and/or DC targeting can cause anti tumor immune response more efficiently in the body, and can more effectively control tumor growth in the animal model (Kretz-Rommel etc., J Immunother 2007,30:715-726).

Except DC, the B cell is the strong antigen-presenting cell of imitating of another type, and it can swash Th1/Th2 cell (Morris etc., J Immunol.1994,152:3777-3785 in advance; Constant, J Immunol.1999 162:5695-5703) and via intersection presents activation cd8 t cell (Heit etc., J Immunol.2004,172:1501-1507; Yan etc., Int Immunol.2005,17:869-773).It is reported that recently (Ding etc., Blood 2008,112:2817-25) in vivo antigen targeted to B cell to be broken the immunologic tolerance of MUC1.

In various embodiments of the present invention, can with to generally incorporating in the immune conjugate vaccine so that with subunit antigen peptide targeted by the APC and the antigenic antibody of expressing by DC especially to immune system cell.Two exemplary APC antigens are HLA-DR and CD74.HLA-DR is the main kind II of histocompatibility complex cell surface receptor, and it works in antigen presentation to cause the T-cell immune response.HLA-DR is found on the multiple antigen-presenting cell, like macrophage, B-cell and BMDC.As stated, the antibody (comprising L243 antibody) to HLA-DR is known in the art.This antibody-like can with the coupling of subunit antigen peptide so that be delivered to APC.

The antigen that another kind of APC expresses is CD74, and it is that suitable MHC II is folding and with MHC II-CD74 complex targeted to the necessary II type of endosome integral protein (Stein etc., Clin Cancer Res.2007,13:5556s-5563s; Matza etc., Trends Immunol.2003,24 (5): 264-8).CD74 expresses and to be not limited to DC, but is found in nearly all antigen-presenting cell (Freudenthal etc., Proc Natl Acad Sci U S A.1990,87:7698-702; Clark etc., J Immunol.1992,148 (11): 3327-35).The wide expression of CD74 in APC can provide and surpass some advantages that just are expressed among the marrow property DC; Because reported the antigen targeted can be broken immunologic tolerance (Ding etc. to other APC (like the B cell); Blood 2008; 112:2817-25), and targeted can antigen be intersected to Plasmacytoid DC and present to CD 8T cell originally.The more important thing is, CD74 also be expressed among the folliculus DC (Clark etc., J Immunol.1992,148 (11): 3327-35), said folliculus DC be the DC subgroup that plays a crucial role for antigen presentation to B cell (Tew etc., Immunol Rev.1997,156:39-52).This express spectra becomes CD74 to be used for the outstanding material standed for of targeting inoculation in the body.Various anti-CD74 antibody are known in the art, like LL1 antibody (Leung etc., Mol Immunol.1995,32:1416-1427; Losman etc., Cancer 1997,80:2660-2666; Stein etc., Blood 2004,104:3705-11).

Antibody and antibody fragment

In various embodiments, antibody or antigen-binding fragments of antibodies can be incorporated in the poxvirus vaccine.Antigen binding antibody fragment is well known in the art, like F (ab ') ₂, F (ab) ₂, Fab ', Fab, Fv, scFv etc., and can use any this type of known fragment.As used herein antigen binding antibody fragment is meant and any fragment complete or the bonded antibody of same antigen that parental antibody is discerned.The technology of almost any target antibody of preparation or segmental conjugate is known (for example United States Patent (USP) the 7,527, No. 787).

Preparation is directed against almost, and the technology of the monoclonal antibody of any target antigen (like HLA-DR or CD74) is well known in the art.Referring to for example Kohler and Milstein, (writing) such as Nature 256:495 (1975) and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, the 1st volume, 2.5.1-2.6.7 page or leaf (John Wiley&Sons 1991).Briefly; Monoclonal antibody can obtain through following steps: will comprise antigenic compositions and inject mice, and take out spleen to obtain the B-lymphocyte, B-lymphocyte and myeloma cell are merged to produce hybridoma; The clone hybridization oncocyte; Select to produce the positive colony of said antigenic antibody, cultivate the clone of the said antigenic antibody of generation and separation antibody from the Hybridoma Cell Culture thing.

Monoclonal antibody (MAb) can be separated and purification from the Hybridoma Cell Culture thing through multiple effective technology.This type of isolation technics comprises affinity chromatography, size exclusion chromatograph and the ion exchange chromatography that utilizes protein

.Referring to for example Coligan 2.7.1-2.7.12 page or leaf and 2.9.1-2.9.3 page or leaf.Also referring to METHODS IN MOLECULAR BIOLOGY such as Baines, the 10th rolls up, and " the Purification of Immunoglobulin G (IgG) " in the 79-104 page or leaf (The Humana Press, Inc.1992).

Grow to after the immunogenic antibody in initial training, but antagonist checks order and prepares through recombinant technique subsequently.The humanization of Mus source antibody and antibody fragment and chimeric be well known to those skilled in the art.Use derives from humanization, antibody component chimeric or people's antibody has been avoided and the relevant potential problems of immunogenicity of Mus source constant region.

Chimeric antibody

Chimeric antibody is the recombiant protein that the variable region of people's antibody is replaced by for example the variable region of mouse antibodies (complementary determining region (CDR) that comprises mouse antibodies).Chimeric antibody presents the immunogenicity of reduction and the stability of increase when using to the experimenter.The general technology in clone immunoglobulin variable territory, Mus source for example is disclosed in Orlandi etc., Proc.Nat ' l Acad.Sci.USA 86:3833 (1989).The technology that makes up chimeric antibody is well known to those skilled in the art.For instance, Leung etc., V through the Mus source LL2 that will encode (a kind of anti--CD22 monoclonal antibody) among the Hybridoma 13:469 (1994) _κAnd V _HThe DNA sequence in territory and corresponding human κ and IgG ₁The constant region domain makes up and produces the LL2 chimera.

Humanized antibody

The technology that produces humanization MAb be well known in the art (referring to for example Jones etc., Nature 321:522 (1986), Riechmann etc.; Nature 332:323 (1988), Verhoeyen etc., Science 239:1534 (1988); Carter etc., Proc.Nat ' l Acad.Sci.USA 89:4285 (1992), Sandhu; Crit.Rev.Biotech.12:437 (1992) and Singer etc., J.Immun.150:2844 (1993)).Chimeric or Mus resource monoclonal antibody can be through coming humanization in the corresponding variable domain that will transfer to people's antibody from the mice CDR of the heavy chain of mouse immuning ball protein and light chain variable chain.The mice framework region (FR) of chimeric mAb is also replaced and is people FR sequence.Because simply with mice CDR transfer to usually cause affinity of antibody to reduce among the people FR or even forfeiture, so possibly need extra modification so that recover original affinity of Mus source antibody.This can realize to obtain to have to the antibody of the good combination affinity of its epi-position through the one or more people's residues in the FR zone being replaced with its Mus source homologue.Referring to for example Tempest etc., Biotechnology 9:266 (1991) and Verhoeyen etc., Science 239:1534 (1988).In general, be different from its Mus source homologue and near or those people FR amino acid residues of adjoining one or more cdr amino acid residues will be the material standed for of replacement.The L243 of humanization form and LL1 antibody are known (referring to United States Patent (USP) for example No. 7612180 and No. 7312318).

People's antibody

The method that the transgenic animal of using combined method or personnel selection immunoglobulin loci to transform produce fully human antibodies is (Mancini etc. for example, 2004, New Microbiol.27:315-28 known in the art; Conrad and Scheller, 2005, Comb.Chem.High Throughput Screen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Phamacol.3:544-50).Fully human antibodies also can pass through gene or chromosome transfection method, and display technique of bacteriophage makes up, and said method and technology all are known in the art.Referring to for example McCafferty etc., Nature 348:552-553 (1990).Compare with chimeric or humanized antibody, estimate that this type of fully human antibodies shows side effect still less and as endogenous people antibody, plays a role basically in vivo.In some embodiments, method of being advocated and the program people's antibody that produces by this type technology capable of using.

In a replacement scheme, display technique of bacteriophage can be used for producing people's antibody (Dantas-Barbosa etc. for example, 2005, Genet.Mol.Res.4:126-40).Can produce people's antibody (Dantas-Barbosa etc., 2005) from normal person or the people who shows particular disease states (like cancer).The advantage that makes up people's antibody from diseased individuals is to make circulating antibody spectrum deflection to the antigenic antibody of disease association.

In a limiting examples of the method, Dantas-Barbosa etc. (2005) make up the phage display library of human Fab's antibody fragment from the osteosarcoma patient.In general, from the blood circulation lymphocyte, obtain total RNA (the same).Reorganization Fab is from μ, γ and κ chain antibody spectrum clone and insertion phage display library (the same).With RNA change cDNA into and be used to use Auele Specific Primer to heavy chain and light chain immunoglobulin sequences produce the FabcDNA library (Marks etc., 1991, J.Mol.Biol.222:581-97).According to Andris-Widhopf etc. carry out library construction (2000, Phage Display Laboratory Manual, Barbas etc. (writing); The 1st edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, the 9.1st to 9.22 page of NY).Final Fab fragment is with digestion with restriction enzyme and insert in the phage genome with the generation phage display library.As known in this area, this type of library can screen through standard phage display method (referring to for example Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart.J.Nucl.Med.43:159-162).

Phage display can be carried out in many ways, about its comment, and referring to for example Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993).People's antibody also can be through producing at external activatory B cell.Referring to United States Patent (USP) No. 5567610 and No. 5229275, its full content is incorporated this paper into way of reference.Those skilled in the art will recognize that these technology are any known methods of manufacturing exemplary and capable of using and screening people's antibody or antibody fragment.

In another replacement scheme, can be used for using the standard immunoassay scheme to produce through genetically engineered transgenic animal and be directed against the antibody of any immunogen target basically with generation people antibody.The method that obtains people's antibody from transgenic mice is by Green etc., Nature Genet.7:13 (1994), and Lonberg etc., Nature 368:856 (1994) and Taylor etc., Int.Immun.6:579 (1994) is open.The limiting examples of this kind system is from Abgenix (Fremont; CA)

(Green etc. for example; 1999, J.Immunol.Methods231:11-23).In

and similar animal; The mouse antibodies gene is through deactivation and replace with the functional human antibody gene, and the remainder of mouse immune system is still intact.

Germ cell line configuration YAC (yeast artificial chromosome) with with the part (comprising most of variable region sequences) that comprises people IgH and Ig kappa gene seat and episome and regulating and controlling sequence transforms.People variable region spectrum can be used to produce antibody and produces the B-cell, and said B-cell can be processed into hybridoma through known technology.

with the target antigen immunity produces people's antibody through normal immunoreaction, and it can standard technique from what has been discussed above be gathered in the crops and/or produce.Multiple

capable of using strain, it can produce different types of antibody separately.People's antibody that the transgenic mode produces has been proved to be has the treatment potentiality, keeps the pharmacokinetic property (Green etc., 1999) of normal person's antibody simultaneously.Those skilled in the art will recognize that compositions and the method advocated are not limited to use

system, but capable of using through genetically engineered any transgenic animal with generation people antibody.

Antibody fragment

The antibody fragment of identification defined epitope can produce through known technology.Antibody fragment is the antigen-binding portion thereof of antibody, like F (ab ') ₂, Fab ', F (ab) ₂, Fab, Fv, sFv etc.F (ab ') ₂Fragment can produce through the pepsin digested antibody molecule, and Fab ' fragment can be through reduction F (ab ') ₂Segmental disulfide bridge bond produces.In addition, can make up Fab ' expression library (Huse etc., 1989, Science 246:1274-1281) has required specific monoclonal Fab ' fragment to allow to identify quickly and easily.F (ab) ₂Fragment can produce through papain digestion antibody, and the Fab fragment can obtain through disulfide bond reduction.

Strand Fv molecule (scFv) comprises VL territory and VH territory.Formation target binding site is united in VL and VH territory.These two territories are further covalently bound through connection peptides (L).The method of making scFv molecule and the suitable connection peptides of design is described in United States Patent (USP) the 4th, 704, No. 692, United States Patent (USP) the 4th; 946; No. 778, R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB the 9th volume: 73-80 (1995) and R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions; " TIBTECH, the 9th volume: 132-137 (1991).

The technology that produces single domain antibody also is known in the art, and is for example disclosed among the Cossins etc. (2006, Prot Express Purif 51:253-259).Single domain antibody (VHH) can for example obtain from camel, alpaca or yamma through the standard immunoassay technology.(referring to for example Muyldermans etc., TIBS 26:230-235,2001; Yau etc., J Immunol Methods 281:161-75,2003; Maass etc., J Immunol Methods 324:13-25,2007).VHH can have the antigen binding capacity of strong effect and can interact to inaccessible new epi-position with conventional VH-VL.(Muyldermans etc., 2001).The alpaca serum IgG contains 50% the only Camelidae heavy chain IgG antibody (HCAb) (Maass etc., 2007) of having an appointment.Alpaca can be used known antigens such as TNF-alpha immunization, and separable combination target antigen and in the VHH (Maass etc., 2007) of target antigen.The PCR primer of nearly all alpaca VHH coded sequence of increasing has obtained identifying and can be used for making up alpaca VHH phage display library that it can be used for coming separation antibody fragment (Maass etc., 2007) through standard biological panning technique well known in the art.

Antibody fragment can prepare through the Proteolytic enzyme of full length antibody or through in another host of escherichia coli or this segmental DNA that encodes, expressing.Antibody fragment can carry out pepsin to full length antibody or papain digestion obtains with conventional method.These methods are described in for example No. the 4th, 036,945, United States Patent (USP) and the 4th, 331, No. 647 and the wherein contained list of references of Goldenberg.Also referring to Nisonoff etc., Arch Biochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959), Edelman etc., METHODS IN ENZYMOLOGY the 1st volume, the 422nd page (Academic Press 1967) and Coligan 2.8.1-2.8.10 page or leaf and 2.10.-2.10.4 page or leaf.

Known antibody

Though the antibody to HLA-DR or CD74 is preferred, poxvirus vaccine alternately uses and combines with lip-deep another antigen of target cell or produce with the antibody of its reaction.Preferred extra MAb can comprise can with humanization, the chimeric or people MAb of CD209 (DC-SIGN), CD34, CD205, TLR2 (toll appearance receptor 2), TLR4, TLR7, TLR9, BDCA-2, BDCA-3 or BDCA-4 reaction.

This antibody-like can be from obtaining like the public source of American Type Culture Collecti or commercial antibody supplier.For example, can be to the antibody of CD209 (DC-SIGN), CD34, BDCA-2, TLR2, TLR4, TLR7 and TLR9 from Santa Cruz Biotechnology company (Santa Cruz, CA) purchase.Antibody to CD205 and BDCA-3 can be from Miltenyi Biotec company (Auburn, CA) purchase.Other numerous antibody commercial source are well known by persons skilled in the art.

These antibody are exemplary and multiple other antibody is known in the art with its hybridoma.Those skilled in the art will recognize that to almost the antibody sequence of any APC-related antigen or the hybridoma of secretory antibody can come simple search ATCC, NCBI and/or USPTO data base to obtain through the antibody to selected associated target antigens.The antigen binding domain of clonal antibody can use standard technique well known in the art to increase, excise, be connected on the expression vector, and transfection is to (adapted) host cell of transforming and be used for protein and produce.

Immune conjugate

In various embodiments, poxvirus vaccine can the immune conjugate administered.Many methods that manufacturing has the covalently or non-covalently conjugate of antibody or fusion rotein are any these type of known methods known in the art and capable of using.

For example, antigenic peptides can be connected in the hinge region through the reduction antibody component via disulfide bond formation.In addition, this type of medicament can use the isodigeranyl functional cross-link agent, connects like N-succinyl 3-(2-pyridine radicals disulfide group) propionic ester (SPDP).Yu etc., Int.J.Cancer 56:244 (1994).This type of link coupled general technology is well known in the art.Referring to for example Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press1991); MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS; Birch etc. (writing); Upeslacis in the 187-230 page or leaf etc., and " Modification of Antibodies by Chemical Methods " (Wiley-Liss, Inc.1995); MONOCLONAL ANTIBODIES:PRODUCTION; ENGINEERING AND CLINICAL APPLICATION; Ritter etc. (writing); Price in the 60-84 page or leaf, " Production and Characterization of Syntheticn Peptide-Derived Antibodies " (Cambridge University Press 1995).

Butt joint and locking (DNL) method

In alternate embodiment, the vaccine based on subunit that comprises immune conjugate can produce through other technology.(DNL) technology is docked and locked to a kind of almost any albumen or peptide being called with any other albumen or the link coupled technology of peptide.The DNL method is utilized in specific proteins/protein-interacting (Baillie etc., the FEBS Letters.2005 that takes place between the grappling territory (AD) of regulation and control (R) subunit and A-kinases anchorin (AKAP) of cAMP-deopendent protein kinase (PKA); 579:3264.Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).

PKA the most completely in signal transduction pathway brings into play central role by second message,second messenger cAMP with the research that combines to be triggered of R subunit a kind of, and it separates (Walsh etc., J.Biol.Chem.1968 first in nineteen sixty-eight from rabbit skeletal muscle; 243:3763).The structure of holoenzyme is formed (Taylor, J.Biol.Chem.1989 by two catalytic subunits that keep inactive form through the R subunit; 264:8443).The R subunit (RI and RII) that the isozyme of PKA comes to light and has two types, and every type have α and β isotype (Scott, Pharmacol.Ther.1991; 50:123).The R subunit is only to stablize that dimeric forms is able to separate and the dimerization territory has been proved to be by preceding 44 n terminal residues and forms (Newlon etc., Nat.Struct.Biol.1999; 6:222).The combining of cAMP and R subunit cause discharging the active catalytic subunit producing the activity of serine/threonine kinases of broad range, it butts up against AKAP through PKA and compartmentation takes place is oriented to selected substrate (Scott etc., J.Biol.Chem.1990; 265; 21561).

Since first kind of AKAP is that MAP-2 obtained characterizing (Lohmann etc., Proc.Natl.Acad.Sci USA.1984 in 1984; 81:6723); Concentrate on various subcellular fractions site and (comprise plasma membrane, actin cytoskeleton, nuclear, mitochondrion; And endoplasmic reticulum) more than 50 kind of AKAP identified has different structure (Wong and Scott, Nat.Rev.Mol.Cell Biol.2004 the species in from yeast to people's scope; 5:959).AKAP is amphipathic helix (Carr etc., the J.Biol.Chem.1991 of 14-18 residue to the AD of PKA; 266:14188).The aminoacid sequence of AD has very big-difference between indivedual AKAP, wherein it is reported to the dimeric binding affinity of RII and arrive (Alto etc., Proc.Natl.Acad.Sci.USA.2003 in the 90nM scope 2; 100:4445).What cause concern is that AKAP only combines with dimer R subunit.For people RII α, AD combines (Colledge and Scott, Trends Cell Biol.1999 with the hydrophobic surface that is formed by 23 n terminal residues; 6:216).Therefore, the dimerization territory of people RII α combines the territory all to be positioned at identical N-terminal 44 aminoacid sequences (Newlon etc., Nat.Struct.Biol.1999 with AKAP; 6:222; Newlon etc., EMBO are J.2001; 20:1651), it is called DDD in this article.

The DDD of people RII α and the AD of AKAP are as the junctional complex module

The AD that we have developed a kind of DDD and AKAP with people RII α as outstanding junctional complex module in case will below be called A and B any two entities to being connected into the platform technology of non-covalent complex, said non-covalent complex can be stablized the structure that bolt is to promote the formation disulfide bond and further be locked as through the strategic location of cysteine residues being introduced DDD and AD.The conventional method of " butt joint-with-locking " approach is following.Entity A is through being connected to the DDD sequence precursor of A, and first component that is called as a below the generation makes up.Because the DDD sequence will realize dimeric spontaneous formation, A will be by a ₂Form.Entity B is through being connected to the AD sequence precursor of B, and second component that is called as b below the generation makes up.a ₂In the dimerization primitive of the DDD that comprised produce with b in the bonded site of docking of AD sequence that comprised, promote a thus ₂With the association at once of b, so that form by a ₂Binary, trimer compositions that b forms.This binding events is owing to coming the subsequent reactions of Covalent Immobilization to become irreversible via disulfide bridge bond two entities; Because making, initial binding interactions place DDD to be in the approximated position so that locus specificity ground is connected with reactive thiol group on the AD; So this follow-up reaction is very efficiently based on the principle of effective local concentration and (Chmura etc., Proc.Natl.Acad.Sci.USA.2001 take place; 98:8480).

In some alternate embodiment, the poxvirus vaccine immune conjugate is based on a ₂The version of b structure, wherein anti--HLA-DR or anti--CD74 antibody or F (ab ') ₂Or F (ab) ₂Each heavy chain of antibody fragment is connected to a copy of AD part at its C-end.Because each antibody or fragment have two heavy chains, so each antibody or fragment have two AD parts.The subunit antigen peptide is connected to complementary DDD part.After DDD part dimerization, each DDD dimer be connected to IgG antibody or F (ab ') ₂Or F (ab) ₂Segmental one of them AD partly combines, and produces each IgG or F (ab ') ₂Or F (ab) ₂The stoichiometric relationship of four antigenic peptides of unit.Yet, those skilled in the art will recognize that alternative composite thing capable of using, as antigenic peptides be connected with the AD sequence with will resist-HLA-DR or anti--CD74MAb or fragment partly be connected with DDD, produces effector different chemical quantitative relation partly.For example, through with DDD sequence and IgG antibody or F (ab ') ₂The C-of segmental each heavy chain is terminal to be connected, and the AD sequence is connected with antigenic peptides, can make up to comprise an antigenic peptides and antibody or segmental DNL complex.

Through DDD is connected away from the functional groups of two precursors with AD, estimate that this type of locus specificity connects the initial activity that can preserve two precursors.The method is modular and can be used for locus specificity ground and the material that covalently is connected broad range potentially in nature.

In preferred embodiments, DDD or AD part is covalently bound to form fusion rotein or peptide with antibody or antigenic peptides.The several different methods of making fusion rotein is known, comprises that nucleic acid synthesizes, hybridizes and/or increases to produce the proteic synthetic double-strandednucleic acid of coding Target Fusion.This type of double-strandednucleic acid can through standard molecular biological technique insert in the expression vector with produce fusion rotein (referring to for example Sambrook etc., Molecular Cloning, A laboratory manual, the 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989).In this type of preferred embodiment, AD and/or DDD part can be connected with the N-end or the C-end of protein or peptide.Yet, those skilled in the art recognize that the connection site of AD or DDD part can change.For example; Though can be when keeping antigen-binding activity that AD or DDD partly are connected to the N-or the C-of antibody or antibody fragment is terminal; But make AD or DDD partly be in terminal being connected of C-and as if produce stronger binding interactions (Chang etc. for example away from the position of antigen binding site; Clin Cancer Res 2007,13:5586s-91s).The locus specificity connection of multiple effector part also can use technology known in the art to carry out, as using bivalence cross-linking reagent and/or other chemical coupling technology.

The method of therapeutic treatment

Preparation

Poxvirus vaccine can be according to the known method preparation with preparation pharmacy suitable compositions, and wherein poxvirus vaccine is with form of mixtures and the combination of pharmacy appropriate excipients.SPBS is an instance of pharmacy appropriate excipients.Other appropriate excipients is well known to those skilled in the art.Referring to for example Ansel etc.; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS; The 5th edition (Lea&Febiger 1990); And Gennaro (writing), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990) and its revised edition.

The preferred subcutaneous or nasal administration of poxvirus vaccine.More preferably, poxvirus vaccine is used via subcutaneous injection with single or a plurality of agent forms of injecting.Administered formulation can provide by unit dosage forms, for example is provided in wherein to add antiseptic in ampoule or the multi-dose container.Compositions can adopt the form as the suspension in oiliness or the aqueous carrier, solution or emulsion, and can contain formula agent, like suspending agent, stabilizing agent and/or dispersant.In addition, active component can be before using the powder type with suitable carrier (for example aseptic apirogen water) rehydration.

Can use other pharmaceutical methods to control the acting duration of poxvirus vaccine.Can prepare slow releasing preparation through the polymer that uses complexation or absorption poxvirus vaccine.For example, bioavailable polymer comprises the acid anhydride copolymer substrate of ethylene-vinyl acetate copolymer substrate and stearic acid dimer and decanedioic acid.Sherwood etc., Bio/Technology 10:1446 (1992).From then on plant the speed that discharges in the substrate and depend on the amount of the molecular weight of poxvirus vaccine, intramatrical poxvirus vaccine and the size of discrete particles.Saltzman etc., Biophys.J.55:163 (1989); Sherwood etc., above-mentioned.Other solid dosage forms is described in Ansel etc.; PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS; The 5th edition (Lea&Febiger 1990); And Gennaro (writing), REMINGTON ' S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company 1990) and its revised edition.

In general, people's dosage of using poxvirus vaccine depends on like factors such as patient age, body weight, height, sex, general medicine situation and medical histories and is different.In single administration, to the receiver about 1mg/kg being provided possibly be desirable to the poxvirus vaccine dosage in the 25mg/kg scope, but as situation needs also can use lower or high dose more.For instance, 1-20mg/kg dosage is 70-1 for 70kg patient, 400mg, or be 41-824mg/m for 1.7-m patient ²But react with induction of immunity like the needs repeat administration.

In alternate embodiment, the treatment peptide can be used (Sievers etc. for example, 2001, Pure Appl.Chem.73:1299-1303) through inhalation route.The supercritical carbon dioxide atomizing has been used for from comprising that the various medicaments of protein and peptide produce the granule (the same) of nanometer or micro-meter scale.With supercritical carbon dioxide and protein or the formed microvesicle of peptide aqueous solution can compare with the alternative method that forms drug powder dry low temperature (25 to 65 ℃) under, thereby keep the structure and the activity (the same) of treating peptide.In some cases, can stabilizing agent such as trehalose, sucrose, other sugar, buffer agent or surfactant be added in the solution, active with further hold function.The granule that produces is small enough to use through suction.In other replacement scheme, the nasal administration of aqueous solution capable of using.

Test kit

Various embodiments can relate to containing and be suitable for treating or the test kit of the component of the illing tissue of diagnosing patients.Exemplary kit can contain at least a or multiple poxvirus vaccine immune conjugate as described herein.Send if comprise the compositions of using component and is not through preparation so that through nasal administration or suck, can come in the device of delivery of agents box component can be included in through subcutaneous injection.One kind of means is the intravital syringe of body that is used for compositions is injected the experimenter.In some embodiments, therapeutic agent can contain the precharging type syringe of aseptic, liquid preparation or lyophilized formulations or automatically injection pen form provide.

Reagent constituents can be packaging together or be separated to two or more containers.In some embodiments, container can be the bottle of aseptic, the lyophilized formulations that contains the compositions that is suitable for rehydration.Test kit also possibly comprise the buffer agent that one or more are suitable for rehydration and/or dilute other reagent.Spendable other container includes but not limited to pouch, dish, box, pipe etc.Reagent constituents can be packaged in the container and keep aseptic.One other component in can being included in is the description that the method for using of test kit is provided to user.

Expression vector

Other embodiment possibly relate to the DNA sequence of the nucleic acid that comprises coding poxvirus vaccine immune conjugate or its constitutive protein.Fusion rotein can comprise the anti--HLA-DR antibody that is connected with the subunit antigen peptide.In addition, coded fusion rotein can comprise DDD or the AD part that is connected with antibody or antigenic peptides.

Various embodiments relate to the expression vector that comprises DNA sequences encoding.Carrier can contain the sequence of light and the CH and the hinge region of the coding human immunoglobulin that can be connected with chimeric, humanization or people's variable region sequences.In addition, carrier possibly contain coded proteinic promoter, enhancer and signal or the targeting sequencing of expression in selected host cell.Useful especially carrier is pdHL2 or GS.More preferably; Light chain and CH and hinge region can be from people EU myeloma immunoglobulins; Wherein randomly at least one aminoacid in allotype (allotype) position is replaced by the aminoacid in the different I gG1 allotype, and wherein randomly replaceable based on the aminoacid of the EU heavy chain of EU numbering system 253 be alanine.Referring to Edelman etc., Proc.Natl.Acad.Sci USA 63:78-85 (1969).In other embodiments, the IgG1 sequence can be exchanged into the IgG4 sequence.

Those skilled in the art will recognize that expression construct is carried out genetically engineered and is inserted in the host cell to express the through engineering approaches method of protein is well known in the art, and belong to routine experiment.Host cell and expression cloning antibody or segmental method for example have been described in United States Patent (USP) the 7531327th, 7537930 and No. 7608425, and the embodiment part is incorporated this paper into way of reference.

Embodiment

Following embodiment is used for explaining and unrestricted claim of the present invention.

Embodiment 1. is to the immunoreation of poxvirus subunit antigen peptide

Material and method

The peptide design. whether the 9-aggressiveness or the 15-mer peptides sequence that have to a plurality of potential binding sites of HLA I class and/or HLA II quasi-molecule are in correct spacing through range estimation examination HLA anchor residues; Or use method based on the WWW (for example BIMAS or SYFPEITHI [Parker etc.; J Immunol 152:163-75,1994; Rammensee etc., Immunogenetics 50:213-19,1999]) and obtain from the poxvirus open reading frame, wherein select to be based on the bonded higher potentiality of specificity HLA-(table 2).VIL18BP (C12L), A4L (Boulanger etc.; J Virol 72:170-79,1998), A27L (Chung etc., J Virol72:1577-85; 1998) or D8L (Hsaio etc., J Virol 73:8750-61) the antigenic nucleotide of VV and aminoacid sequence go into to hide registration number from NIH GenBank: AY243312Retrieve.These peptides are named through its gene source and numbering (for example vIL18BP105 or vD8L118).

The amino acid number of table 2. poxvirus vIL18BP-derived peptide, A4L229 and TT830 and HLA restriction.

* TT830, tetanus toxoid T cell is assisted peptide; ^#A4L229 is from the epi-position of cowpox A4LORF; VIL18BP, the conjugated protein derived peptide of poxvirus IL-18 (cowpox C12L). do not have combination potentiality>=15 (www.SYNPEITHI.de) of round parentheses; In round parentheses, join probability is 10-14.Except HLA II class epi-position, the 15-aggressiveness also can contain potential HLAI class combination epi-position more than.The HLA type that only shows limited quantity.

The peptide ammino acid sequence is as follows.Similarity, use NIHBlast service routine (http://www.ncbi.nlm.nih.gov/BLAST/) to the people's gene group screen peptide.The peptide that has with human protein group homology is dropped.All newly-designed peptides with 95% purity come commercial synthetic (Sigma-Genosys, Woodlands, TX USA, New England Peptide, Gardner, MA).

vIL18BP118

CVLTTLNGV(SEQ?ID?NO：13)

vIL18BP102

KFAHYRFTCVLTTLNGVSKKNIVVLK(SEQ?ID?NO：14)

vIL18BP105

HYRFTCVLTTLNGVS(SEQ?ID?NO：15)

vIL18BP110

CVLTTLNGV(SEQ?ID?NO：16)

vIL18BP117

GVSKKNIWL(SEQ?ID?NO：17)

VA4L229 (smallpox virus)

ALKDLMSSV(SEQ?ID?NO：18)

TT830 (clostridium tetani)

QYIKANAKFIGITEL(SEQ?ID?NO：19)

vA27L003-027

GTLFPGDDDLAIPAT(SEQ?ID?NO：20)

vA27L003-012

GTLFPGDDDLAIPATEFFSTKAAKK(SEQ?ID?NO：28)

vA27L004-012

TLFPGDDDL(SEQ?ID?NO：29)

vD8L110-134

HDDGLIIISIFLQVLDHKNVYFQKI(SEQ?ID?NO：21)

vD8L118-132

SIFLQVLDHKNVYFQ(SEQ?ID?NO：22)

vD8L116-124

IISIFLQVL(SEQ?ID?NO：33)

vB5R001-025

MKTISVVTLLCVLPAVVYSTCTVPT(SEQ?ID?NO：30)

vB5R004-018

ISVVTLLCVLPAVVY(SEQ?ID?NO：31)

vB5R008-016

TLLCVLPAV(SEQ?ID?NO：32)

The donor sampleBlood Center (NJBB) (West Orange, NJ USA) obtains buffy coat from the New Jersey.Other PBMC sample is ratified end user's blood afterwards from local donor in New England's institutional review board (Wellesley, MA USA), or obtains from Cellular Technology Limited (CTL) (Shaker Hts, OH USA).Table 3 has been summed up donor HLA type, age and vaccine state.Because the cell quantity in each sample is limited, not every sample all is included in each test.

Gathering of table 3. blood donor vaccine state, age and HLA type.

(Germany) description increases for Biotest, Dreieich according to the HLA-parting kit from the DNA of donor PBMC.Be CTL, the Inc. sample provides HLA type.The inoculation donor is to state it once to accept the people of antismallpox vaccine alive, perhaps infers the inoculation state according to the age, is to state it not accept the people of variolation or birth after the U.S. stops to inoculate and do not inoculate donor.Because the cell quantity in most of samples is limited, not every sample is all tested in all tests.When the HLA of donor type was not confirmed by supplier, (Biotest, Dreieich Germany) measured the HLA type of PBMC through SSP-PCR to use the Biotest test kit.

Peptide screeningWith antigen processing albumen-1 and-2 (TAP1 and 2)-defect type human B/T hybridoma cell line T2 cell (ATCC; Manassas; VA USA) surperficial HLA-A02 is only expressed in relevant transport protein, and in the antigen presentation passage, can increase its expression (Nijman etc., Eur J Immunol 23:1215-19 during by stabilized peptide; 1993), beta-2-microglobulin and the peptide of this transport protein under indication concentration hatched.Because without processing, therefore must having, TAP defective, peptide allow and the bonded length of HLA-A02 (9-aggressiveness).Use the W6/32 (BD Pharmingen, San Diego, CA USA) and the FACSCALIBURTM flow cytometer (Becton Dickinson, San Jose, CA USA) of FITC-labelling to carry out the HLA analysis.Through with 1 * 10 ⁶The PBMC of/mL is hatched with the biotinylation peptide, then avidin-FITC conjugate is added into fixed cell and carries out flow cytometry, comes combining of detection of peptides epi-position and the human PBMC who obtains from donor.

T-cell proliferation and phenotype analyticalIn order to estimate; Use is based on CF 5(6)-Carboxyfluorescein diacetate succinimide ester (Invitrogen; Carlsbad, CA USA) cell proliferation test (Younes etc., J Exp Med 198:1909-22; 2003), external to from inoculation smallpox and originally the PBMC of donor carry out peptide screening.For the ease of relatively, will derive from immundominance poxvirus protein A 4L (Boulanger etc., J Virol 72:170-79; 1998) peptide, another is from tetanus toxoid (TT830) (Demotz etc.; J Immunol 142:394-402,1989) peptide, or from HIVgag albumen (HIVgag) (Kan-Mitchell etc.; J Immunol172:5249-61,2010) in peptide is included in.Briefly, with 10-50 * 10 ⁶PBMC is with CFSE (1.5 μ M) labelling.2 * 10 ⁵The peptide, Staphylococcus aureus enterotoxin (SEA) of cell (200 μ L) and indication concentration (10ng/mL) or phytohemagglutinin (2.5 μ g/mL) (PHA Sigma-Aldrich) is hatched together.Cell is used the antibody staining to CD8 or CD3, and after 5 days, checks viability (7-AAD).To collect with flow cytometry with 20,000 incidents that the CD3+ lymphocyte of living is established door, and (Mountain View CAUSA) analyzes to use Flow-Jo software.Minimizing based on CFSE fluorescence comes assessment of proliferation.Number number of proliferating cells when not having peptide (contrast) that will when having stimulator polypeptide (test), lose the cell of CFSE dyestuff calculates the fluorescence index (FI) of proliferative cell.

For phenotype analytical, the PBMC among the GOLGIPLUGTM (brefeldin A, 1 μ g/mL) is hatched 14h with 10 μ g/mL monitor peptides, culture medium contrast (wherein adding the PBS with peptide storing solution equal volume) or SEA or PHA.Then with cell (after permeability) with indicated FLA (IFN-γ or IL-2) in the surface or in cell inner dyeing.Also whether cell is contacted antigenic CD45RA, CCR7 (lymph node go back to the nest mark) (38) or CD107a (cell killing capability flags thing) (1) dyeing to CD8, before being used for confirming.In peptide stimulation and controlled trial, measure CD8+ or CD8-effect memory (T _EM) or last eventually T cell (CD45RA-CCR7-), the central memory T cell (T of breaking up _CM) (CD45RA-CCR7+) drive differentiation T cell (T with cytokine _EMRA) (CD45RA+CCR7-) percentage ratio of (12).The negative T cell of CD8-is considered to contain CD4+ colony.

Antibody analysisUse is assessed serum antibody based on the modification method (Makabi-Panzu etc., Vaccine16:1504-10,1998) of ELISA.Briefly, 10 μ g/mL target peptides are coated in the elisa plate hole.After blocking-up and washing, be added on the test sera of 2 times of serial dilutions among the PBS.To detect and the combining of antibody with the link coupled anti-people's antibody of peroxidase.Plate is developed with o-phenylendiamine dihydrochloride peroxidase substrate (Sigma-Aldrich, St.Louis, MO USA) and with the optical density of ELIASA measured hole under 490nm.

Data analysisThe observed significance of difference is used Statview+SE software (Abacus Concepts by indication under experiment condition; Berkeley; CA USA) or variance analysis (ANOVA) (Excel; Microsoft Corp., Redmond, WA USA) proofread and correct to confirm through StudentShi t check and the Fischer that accompanies by multiple comparisons.It is significant that P＜0.05 is considered to.

The result

Design of pox phallotoxins and screeningAccording to SYFPEITHI or BIMAS hierarchy system, combine the higher score of potentiality, poxvirus vIL18BP (SEQ ID NO:23) divided classify 9-, 15-or 25-mer peptides as, focus on HLA-A02-and HLA-DR04-combination based on HLA-.Test vIL18BP-derived peptide combines with TAP-deficiency T2 hybridoma, and it is the expression of increase HLA-A02 during by stabilized peptide in the antigen presentation passage.9-mer peptides, vIL18BP110 (SEQ ID NO:16), vIL18BP117 (SEQ ID NO:17) and A4L (SEQ ID NO:18) contain the sequence (without processing) with potential HLA-A0201 binding ability.In these peptides, vA4L (SEQ ID NO:18) and vIL18BP110 (SEQ ID NO:16) combine the HLA-A02 (Figure 1A) on the T2 cell with the mode of concentration dependent.VIL18BP117 (SEQ ID NO:17) arrives the height probability although peptide has the moderate that combines HLA-A02, combines.And there is the 15-mer peptides (vIL18BP008, SEQ ID NO:13 and 105, SEQ ID NO:15) of the vIL18BP110 sequence (SEQ ID NO:16) that the T2 cell can not process also not combine.

The vIL18BP sequence

MRILFLIAFMYGCVHSYVNAVETKCPNLDIVTSSGEFYCSGCVEHMSKFSYMYWLAKDMKSDEYTKFIEHLGDGIKEDETIRTTDGGITTLRKVLHVTDTNKFAHYRFTCVLTTLNGVSKKNIWLK(SEQ?ID?NO：23)

Predict that also the vIL18BP peptide can combine some other HLA haplotypes (table 2), wherein great majority are presented in the PBMC donor colony of general introduction in the table 3.When the combining of test peptides and donorcells, vIL18BP008 (SEQ ID NO:13,15-aggressiveness) and vIL18BP110 (SEQ ID NO:16; The 9-aggressiveness) represent with from donor NJ04 (A01/03; DR04) and NJ01 (A11, the strong combination of PBMC DR15), and with NJ07 (A01; DR16) and NJ08 (A01/02, weak relatively combine (Figure 1B) of PBMC DR16).Consider the prediction HLA target and the T2 result of donor HLA-type, peptide; Can reach a conclusion: vIL18BP110 (SEQ ID NO:16) 9-aggressiveness does not combine HLA-A01; But combine HLA-A02, A03 and-A11, it is all represented by T2 cell or donor group.

15-aggressiveness vIL18BP105 (SEQ ID NO:15) it is predicted all the I class HLA types except that HLA-A01 that can combine HLA II class DR04 and DR15 (NJ01 and NJ04) and represented by donor.Except HLA-A01, donor NJ08 also is that HLA-A02-is male, so HLA-A02 can explain measured adhesion.Strong signal from the donor NJ07 of expression of HLA-DR 16 and non-binding HLA-A01 has hinted vIL18BP105 (SEQ ID NO:15) and the bonded evidence of HLA-DR16.Though the total primitive of HLA-DR16 is never characterized (Onion etc. preferably; J Gen Virol 88:2417-25,2007), but the combination primitive share similar property (Zeng etc. of at least one report hint HLA-DR15 and-DR16 are arranged; J Virol 70:3108-17,1996).

Immunoreactivity as the peptide of T cellular antigens analogies was assessed based on the proliferation test of CFSE through 5 days, wherein will hatch with vIL18BP105 (SEQ ID NO:15) with from the peptide of two kinds of other poxvirus genome vD8L118 (SEQ ID NO:22) and vA27L003 (SEQ ID NO:20) from inoculation or the PBMC that does not inoculate the load C FSE of donor.The result of all vIL18BP105 (SEQ ID NO:15) test is summarized in the table 4.The result of the parallel test of vIL18BP105 (SEQ ID NO:15), vD8L118 (SEQ ID NO:22) and vA27L003 (SEQ ID NO:20) is shown in Fig. 2 A (inoculation donor) and Fig. 2 B (not inoculating donor).

Generally, vIL18BP105 (SEQ ID NO:15) induces the remarkable propagation (table 4) from the PBMC of inoculation donor with the concentration of 10 μ g/mL.With vD8L118 (SEQ ID NO:22) (6/7) and vA27L003 (SEQ ID NO:20) (4/7, Fig. 2 A) when hatching together, the cell of inoculation donor is also bred.These results show that vIL18BP105 (SEQ ID NO:15), vD8L118 (SEQ ID NO:22) and vA27L003 (SEQ ID NO:20) comprise the epi-position of discerning by from the lymphocyte of variolation donor.From the cell of not inoculating donor generally for pox phallotoxins reactionless (Fig. 2 B).When in independent experiment (14-h test), detecting when whether having the activation mark and producing the cell within a cell factor discovery IFN-gamma reaction in CD4+ and CD8+ cell from inoculation donor (12A, 416,417) and the sample of not inoculating donor (213,704,706).The CD8+ cell is express cell kill capability mark CD107a (than culture medium contrast, P＜0.05, Fig. 2 C) also.

Table 4. is for the summary sheet of the PBMC breeder reaction of vIL18BP105

Use 10 μ g/mL vIL18BP105 peptides, 2.5 μ g/mL PHA (P) or 10ng/mLSEA (S) concentration.The cell proliferation test that use is described in material and method based on CFSE, through will be when having peptide (or P or S) number number of proliferating cells when not having peptide (or P or S) of proliferating cells estimate fluorescence index (FI).The number of the individual donor of N=.All samples detects (meansigma methods ± SD) three times.For vIL18BP105, the inoculator is than inoculator not, P＜0.001 (ANOVA).

The phenotype of proliferative cellFor CD4 or the CD8 phenotype of measuring proliferative cell, detect with what vA27L003 (SEQ ID NO:20) or vIL18BP105 (SEQ ID NO:15) were hatched (5 days) whether express CD4 or CD8 with the PBMC of the load C FSE of inoculation contrast from inoculation.In the sample from the inoculation donor, CD4+ (4/5) and CD8+ (2/5) cell are all bred, and in not inoculating the sample of donor, arbitrary cell subsets is not all almost bred (0/3, table 5).

In the dye test of the 14-hour cell inner cell factor, carry out the further mensuration of responsive cell phenotype.In the sample of hatching, find the increase (table 6, P＜0.05) that the IFN-γ in the CD8+T cell colony produces with vD8L118 (SEQ ID NO:22) (2/5) or vIL18BP105 (SEQ IDNO:15) (2/5).Although 5 days the test moderate stimulation propagation based on CFSE, vA27L003 (SEQ ID NO:20) did not stimulate IFN-γ or IL-2 to increase (not shown).In the CD4+T cell, IFN-γ does not produce significantly to be increased, but sample separation is reacted.Yet in CD4+ colony (vD8L118, SEQ ID NO:22) and CD8+ cell (vIL18BP105, SEQ ID NO:15), IL-2 produces increases significantly (table 6; P＜0.04).

The CD4+ of the proliferative T cell that table 5 is hatched with vA27L003 or vIL18BP105 peptide or CD8+ phenotype (test in 5 days).

The human PBMC who deposits is thawed, hatch 24h, then hatch with PHA-P (2.5 μ g/mL) or 20 μ g/mL peptides, dye afterwards and express to detect CD4 or CD8 by indication with CFSE.Pass through flow cytometry.Column headings: continued: culture medium contrast; VA27L:vA27L003; VIL18BP:vIL18BP105.The value that appears with boldface type is than >=1.5 times of contrasts.

Through being directed against originally cd8 cell and effector cd8 cell subgroup (T _EMRA) mark CD45RA, dye to come further to analyze T cell from the generation CD8+/IFN-γ of identical inoculation donor and whether have the mark with the memory phenotypic correlation with the lymph node mark CCR7 that goes back to the nest.This analyzes T _CM(CCR7+CD45RA-), precursor (CCR7+CD45RA+), T _EMRA(CCR7-CD45RA+) and T _EMDistinguish with whole end differentiation (CCR7-CD45RA-) cell colony.The cell type that develops be CCR7-CD45RA-(TEM or end differentiation effector) eventually (Fig. 3).VD8L118 (SEQ ID NO:22) antigenic peptides the most active in producing these cell types (than culture medium contrast, P＜0.019).In addition, 2 donors in each test are also made similar reaction to vIL18BP105 (SEQ ID NO:15) with vA27L003 (SEQ ID NO:20).

Table 6. hatches the CD4 of 14h with the pox phallotoxins and the IL-2 or the IFN-γ of cd8 t cell produces.

Institute's indicating value is the percentage rate about the positive cell of every kind of cytokine of hatching each donor after the 14h with specified polypeptide.Cell is dyeed to CD8, and the negative lymphocyte of CD8-is considered to CD4+.The CD8+ cell is made up of the total lymphocyte of 10-30%.All donor Smallpox vaccinations.Peptide concentration: 10 μ g/mL; PHA, 2.5 μ g/mL.The value that appears with boldface type surpasses >=1.5 times of culture medium contrasts.Reaction for HIV peptide and vA27L003 is not significantly to be different from the contrast that culture medium is only arranged.* CD8/IFN-γ/vD8L118 and vIL18BP105 contrast than culture medium, P＜0.05; CD4/IL-2/vD8l118 contrasts than culture medium, P＜0.04; CD4/IL-2/vIL18BP105 contrasts than culture medium, P＜0.013 (t-check).

Detect the ability of CD8+ effector lymphocyte colony threshing through the expression of measuring CD107a, promptly its ability (Berhanu etc., J Virol 82:3517-29,2008) of carrying out effector function (Fig. 4).CD8+IFN-γ+with CD8+IFN-γ-colony in, in 3 PBMC samples in 5 PBMC samples of hatching with vD8L118 (SEQ ID NO:22), CD107a expresses and to be increased to 2-7 doubly (P＜0.04).When hatching, also in CD8+IL-2+ colony, measuring CD107a increases with vIL18BP105 (SEQ ID NO:15) (P＜0.01) and vD8L118 (SEQ ID NO:22), but the latter does not obtain significance.

In similar 14-hour cell inner cell factor dye test, from CD8+IFN-γ+cell of not inoculating donor for peptide reactionless (not shown).

Serum antibody titerNeeds are directed against the antibody of poxvirus so that protection is provided when secondary exposes, and are inoculating decades afterwards, and the existence of anti-vaccinia antibody still remains in 90% receive (Hammarlund etc., Nat Med 9:1131-37,2003) among the inoculator.Therefore, the serum antibody from the patient who inoculated in the past will point to the relevant B cell epitope of immunity.Whether comprise discernible B cell epitope in order to measure antigenic peptide sequence, will test with peptide vA27L003 (SEQ ID NO:20) (15-aggressiveness), vIL18BP102 and vD8L110 (25-aggressiveness) from inoculation and 1: 200 dilute serum not inoculating donor.Result (Fig. 5) shows to the serum antibody of vD8L110 and vA27L003 (SEQ ID NO:20) peptide higher generally, and is significantly higher than not inoculation individual (P＜0.05).Though 3 donors produce the antibody of identification vIL18BP102, total result does not obtain significance.The result shows that experimental peptide contains one or several sequence of serving as the B cell epitope.The existence of anti-peptide antibody is not because donor or postvaccinal time and different (not shown).

Discuss

Antigenic peptides is included in substitutes that to require them in the orthopoxvirus vaccine are relevant targets of body immunity.Whether the The above results determining source can cause anamnestic response from the antigenic particular peptide of poxvirus in the PBMC of inoculation donor.Epi-position derives from three vaccination virus antigens; Be included in the antigen (vIL18BP) that host immune power aspect characterizes; And characterize to host protection; But known poxvirus envelope antigen A27L that its defined epitope characterizes and D8L (Chung etc., J Virol 72:1577-85,1998; Hsaio etc., J Virol 73:8750-61,1999).The response type that is caused by every kind of peptide is different.

Poxvirus IL18BP is through neutralizing NK cell IL-18, and then prevents that IFN-γ from producing, and comes host's innate immunity is regulated.CD4+ and CD8+ cell and confirmed following hypothesis for one group of peptide vIL18BP, vIL18BP105 (SEQ ID NO:15) and the identification of its derivant from the serum antibody of inoculation donor: it is the target of host immune power that virus host this and other transient expression probably reacts regulatory factor.The infection foundation that the neutralization of vIL18BP can help to prevent primary infection and cause thus, this conclusion is recently by conjugated protein proof of poxvirus type I IFN-(Xu etc., J Exp Med 205:981-92,2008).

Design demand virus component and HLA based on the vaccine strategy of epitope interact so that the T-cell development.In this research, peptide it is predicted and can be combined some HLA haplotypes that defined.But, although exist about the bonded prediction of HLA, such as from the peptide of C12L sequence (vIL18BP) proof, have only some epi-positions to combine.

Peptide from C12L (vIL18BP) and antigen A 27L and D8L causes from the CD4+ of inoculation donor and the propagation of CD8+ cell; This show that APC absorbs peptide and under the situation of HLA I class with its processing being presented to CD8+, and under the situation of II class with its processing to be presented to the CD4+ lymphocyte.

Immunity to poxvirus depends on cellular immunization and humoral immunization (Dunne etc., Blood 100:933-40,2002; Ferrier-Rembert etc., Viral Immunol 20:214-20,2007; Meseda etc., Clin Vaccine Immunol 16:1261-71,2009; Xu etc., J Immunol 172:6265-71,2004), the two needs auxiliary classification conversion and the affinity maturation realized of T cell.The proximity of T and B epi-position possibly influence the antibody generation owing to its difference of presenting in polypeptide.Yet the natural B cell epitope is often near HLA-calmodulin binding domain CaM (Simitsek etc., J Exp Med 181:1957-63,1995).Many factors possibly influence the formation of antibody, comprise HLA type (Quaratino etc., J Immunol 174:557-63,2005).

In aforesaid research, we prove in the 1/3 inoculation donor subgroup and measurable antibody occurs.In addition, peptide comprises linear order, and therefore antibody recognition linear epitope, and it can comprise only 3 aminoacid (Tahtinen etc., Virology 187:156-64,1992).Under the situation of vIL18BP105 (SEQ ID NO:15); Exist the further evidence of relevant B cell epitope to provide, in said research, discern total length reorganization vIL18BP (C12L) protein (not shown) to the serum antibody of vIL18BP105 (SEQ IDNO:15) by mice study.Generally speaking, these results show that the antigenic peptides of in this research, using is presented the T-and the B-cell target of people's reaction.

The generation of cytokine and mark has shown that vIL18BP105 (SEQ ID NO:15) and vD8L118 (SEQ ID NO:22) cause the generation of IL-2; It has preserved the multiplication capacity of T cell; Even also be down (Zimmerli etc. like this there not being CD4 auxiliary; Proc Natl Acad Sci USA 102:7239-44,2005).This has supported the propagation data, and has proved that further the vIL18BP peptide is for the effectiveness based on the vaccine of subunit.The IFN-γ of the CD8+TEM cell that peptide stimulates produces has multiple-effect, comprises inducing anti-disease toxic effect function.Therefore, when vD8L118 (SEQ ID NO:22) stimulation had the CD8+T cell of effect and multiplication potentiality, the result was similar to for the cell institute results reported of hatching with virus (Laouar etc., Plos One 3:e4089,2008).IFN-γ produces also becomes the distinctive feature that produces the Th1 reaction, and said Th1 reaction is essential (Meseda etc., Clin Vaccine Immunol 16:1261-71,2009 for the cytotoxicity that occurs to cowpox; Xu etc., J Immunol 172:6265-71,2004).In addition, participate in closely linking together by the IL-2 of CD4+ cell generation and the T-cell of IFN-γ and auxiliary Th1 guiding.The A27L peptide does not stimulate IFN-γ or the IL-2 in CD8+ or the CD4+ cell to increase, and is like this even the T cell is able to propagation when peptide is hatched together therewith yet.Activity in the A27L sample lacks maybe be owing to different kineticses, or the alternative cell colony that is stimulated produced us and not have the different cytokines or the interleukin of detection, like IL-4.

Our result about cell killing capability flags thing CD107a is similar to a research recently; Wherein stimulating the back to express CD107a (Puissant-Lubrano etc. with vaccinia virus from the CD4+ cell subsets that inoculates donor; J Clin Invest 120:1636-44,2010).In our research, the enhanced expression that after stimulating, shows this mark from the CD8+ cell of inoculation donor with peptide.

Opposite with the discovery of Combadiere etc. (J Exp Med 199:1585-89,2004), when variola vaccination betide surpass 45 years before the time, we do not observe the forfeiture that produces the ability of cytokine in response to cowpox antigen.

In a word, the evidence that we have proposed shows, can stimulate the anamnesis reaction of inoculation donor from the subunit antigen peptide of 3 vaccination virus antigens, comprises the identification for the B cell epitope of T cell proliferation, cytokine expression and serum antibody.A kind of host defense regulatory factor IL18BP who characterizes so far that epitope comes free cowpox to produce.Here the result who provides shows that the alternative vaccine that uses selected peptide epitopes to develop to poxvirus can produce immunity, and challenge virus of no use is inoculated the harm that is brought.With respect to Disease-causing gene usually the poxvirus of the attenuation form of disappearance or sudden change carry out immunity, in the advantage of this virus-free method is that the gene of immune relevant portion and the change of any poxvirus genome can be included in.

Embodiment 2.APC-targeting antibodies and the coupling of subunit antigen peptide are to be used for poxvirus vaccine

General introduction

VIL18BP105 (SEQ ID NO:15) and anti--HLA-DR antibody L243 coupling so that be presented to immune system better, and are used its transgenic that makes expression of HLA-DR 04 (tg) mouse immune.Compare with free vIL18BP105 (SEQ ID NO:15), coupling vIL18BP105 (CIL18BP105) is absorbed by people and HLA-DR transgenic mice cell more easily.The splenocyte of HLA-DR04 transgenic mice of CIL18BP105 immunity of using by oneself is bred when external use vIL18BP105 (SEQ ID NO:15) stimulates.The propagation of the mouse boosting cell of CIL18BP105 inoculation relates to the CD3+CD4+CD45RA cell.Propagation produces (quantitatively sandwich ELISA) with interferon gamma.Booster immunization is 7 days afterwards for the first time, and the mice of inoculation CIL18BP105 also demonstrates 4 times of shifting to an earlier date and zooming peptide specific antibody titer to the contrast of injecting vIL18BP105.In the time after a while, CIL18BP105 and vIL18BP105 (SEQID NO:15) induce IgG2a and IgG1, and this prompting Th1 and Th2 immunity are initial.Discern complete recombinant C 12L albumen from the serum antibody of CIL18BP105 immune mouse.These results show that immunogenicity has been strengthened in the coupling of antigenic peptides and anti-HLA-DR antibody, and have promoted the delivery of peptides of the antigen-presenting cell of expression of HLA-DR.

Method

HLA-DR antibody-conjugateUse contains the isodigeranyl functional cross-link agent of H-HOSu NHS (NHS) ester and maleimide base group; Sulfosuccinimide base-4-(N-maleimide ylmethyl) thiacyclohexane-1-carboxylic acid (sulfo group-SMCC) " SMCC "; Follow the scheme (Pierce of manufacturer; Rockford, IL, but the peptide and the L243 antibody coupling that USA) immune stimulatory donor PBMC are bred.SMCC interacts with the formation amido link through the primary amine of its NHS ester group and antibody, and the free sulfhydryl groups of the C-terminal cysteine on maleimide base group and the peptide forms thioester bond.Briefly, the solution of SMCC in PBS of the 1mg/ml prepared fresh of solution and the 20 μ Ls of the 1mg/ml antibody of 1ml in PBS is 4 ℃ of reactions 2 hours down.After hatching, unnecessary SMCC is removed through PBS pre-equilibration desalting column.Activation antibody is collected, then 4 ℃ down and peptide (having the C-terminal cysteine) hatch 2 hours (or spending the night) again.The thin crosslinked pearl post of P60 is got rid of, used to conjugate through volume, and (CA USA) comes purification to remove free peptide for BioRad, Hercules.Use the gradient gel of 5%-20% to come the evaluation of peptide coupling efficiency through SDS-PAGE.Before injecting mice, the conjugate preparation is through 0.22-μ m PVDF filter (Millipore, Bedford, MA) filter sterilization, and emulsifying in incomplete Freund's adjuvant (IFA).

Mouse immuneFrom Taconic (Germantown, NY, USA) six to eight of acquisition expression of HLA-DR 04 (HLA-DR tg) all female C57BL/6J in age (B6) transgenic (tg) mices.Mice remains in the pathogen-free domestic zone of our facility.In order to carry out immunity, each groups of every group of 3 mices is swashed in advance, then through subcutaneous route, with 25 μ g to dissociate or antibody coupling form emulsive vIL18BP105 (SEQ ID NO:15) peptide in IFA serves as that the interval is strengthened twice with two weeks.The mice of injection IFA emulsifying PBS serves as contrast originally.From swashing in advance to putting to death, serves as to collect blood at interval with a week, said execution is after the booster immunization 7 days the last time.When putting to death, collect the spleen sample.Be used under 4 ℃, spending the night and prepare from blood after the blood coagulation through the serum that ELISA carries out antibody test and typing.Separating by the stainless steel cloth mechanical damage through making spleen channel based on the splenocyte that uses in the T-cell proliferation test of CFSE and the TCR analysis of spectrum.

Antibody produces to be analyzed and typing mensurationThe modification method based on ELISA (MakabiPanzu etc., 1998) of report is assessed production of antibodies and homotype before using.Briefly, with the elisa plate hole coat among the PBS 10 μ g/ml peptides and 4 ℃ of following incubated overnight.Blocked 30 minutes down and wash at 37 ℃ with skimmed milk/PBS then with the PBS (PBST) that contains 0.05% polysorbas20.The test sera of adding 2 times of serial dilutions to be obtaining antibody titer, or measures to be used for homotype with dilution in 1: 200.At room temperature plate and serum are hatched 2h.With unnecessary serum through removing for three times with PBST washing, and at room temperature add dilution in 1: 1000 last 45min with the link coupled goat anti-mice of peroxidase (or under the situation of typing with the link coupled sheep anti of peroxidase-mice IgA, IgG1, IgG2a, IgG2b, IgG3 and IgM).After this hatches, with the hole washing, add peroxidase substrate, and after developing, with the OD of ELIASA measured hole under 490nm.

T-cell proliferation test and TCRV β analysis of spectrumThe T-cell proliferation of donor PBMC or Mus source splenocyte used like former report and assessed (Younes etc., 2003) based on the cell proliferation test of CFSE in 5 days.Briefly, 10-50 * 10 ⁶PBMC or splenocyte under 1.5 μ M ultimate densities with the CFSE labelling.With cell washed twice in PBS, and be suspended in again in the complete RPMI culture medium with 106 cells/ml.With 2 * 10 ⁵Cell is hatched to obtain the positive control hole with the peptide or the PHA (2.5 μ g/ml) of indicated concentration.External at 37 ℃, 5%CO ₂After hatching in the atmosphere 5 days, cell is dyeed with CD4-APC, CD8-PE and 7-AAD or CD3-perCp.Minimum 20,000 incidents of establishing door with the CD3+ lymphocyte of living are able to collect on the FACScalibur flow cytometer, and use Flow Jo software to analyze.Reduce based on the CFSE fluorescence of auxocyte and to estimate the T-cell proliferation.Comprehensive cell proliferation Flow Jo program is used for analyzing.Number number of proliferating cells when not having peptide (contrast) that will when having stimulator polypeptide (test), lose the cell of CFSE dyestuff calculates the fluorescence index (FI) of proliferative cell.

For TCRV β analysis of spectrum; To wash with complete RPMI-1640 culture medium and dyeing buffer once more from warp washing splenocyte immune or mice originally; Then as stated to the T-cell surface marker at 4 ℃ of following pre-staining 20min, and then under 4 ℃ with blocking-up 2.4G2 anti--FcRIII/I mAb hatches 15min.Cell resists-TCRV β antibody staining with suitable fluorescently-labeled then, and does not remove FcR-blocking-up mAb.After this hatches at last, cell is washed and uses flow cytometry with the dyeing buffer.

Data analysisThe observed significance of difference is used Statview+SE software (Abacus Concepts, Berkeley, CA USA), is used one way analysis of variance under experiment condition, then t check and the Fischer that accompanies by multiple comparisons are proofreaied and correct to confirm according to circumstances.It is significant that P＜0.05 is considered to.

The result

At external T-cell proliferation in response to vIL18BP105 (SEQ ID NO:15) peptideWhether the coupling of subunit antigen and APC targeting mAb will produce the enhance immunity reaction in order to test, with peptide vIL18BP105 (SEQ ID NO:15) with mAb L243 (CIL18BP105) chemical coupling and use it to make mouse immune.The result is compared with the mice of free vIL18BP105 (SEQ ID NO:15) in accepting PBS/IFA (originally) and IFA.Use the body outer cell proliferation test based on CFSE in 5 days, breed with the concentration dependent mode from the splenocyte of CIL18BP105 inoculation mice.From originally or the cell of free peptide immune mouse reactionless relatively (Fig. 6).TCRV β spectrum deflection TCRV β 8.3 (not shown) of the positive splenocyte of CD4-of the HLA-DR04tg mice after the vIL18BP105 of arbitrary form of hanging oneself (the SEQ ID NO:15) immunity.

Antibody in response to vIL18BP105 (SEQ ID NO:15) peptide producesHumoral immunization to poxvirus is absolutely necessary for protecting from infection.Therefore, in immune HLA-DR04tg mice, studied the antibody response than vIL18BP105 (SEQ ID NO:15) to CIL18BP105.The result is shown among Fig. 7.Compare with the mice of injecting vIL18BP105 (SEQ ID NO:15), the 7th day (swashing in advance the 21st day afterwards) after the booster immunization for the first time, the mice of injection CIL18BP105 shows that higher peptide specific antibody produces.But after first time booster immunization 14 days, the quantity of antibody was similar.IgG1 and IgG2a homotype all can be induced by CIL18BP105 and vIL18BP105 (SEQ ID NO:15), but compare with its free homologue, and CIL18BP105 causes that more IgG1 antibody produces (not shown).The generation of IgG1 and IgG2a has hinted the ripe antibody response under the T cell is auxiliary.This antibody response has also hinted Th1 and the participation of Th2 accessory cell.From the serum antibody and whole vIL18BP albumen (C12L) kickback (Fig. 8) of CIL18BP105 immune mouse, show with the subunit antigen peptide of anti--APC antibody coupling and can induce systemic immune response to complete virus particle.Produce aspect the interferon-at the external splenocyte that stimulates through vIL18BP105 (SEQ ID NO:15) peptide promoting, with the CIL18BP105 immunity than with vIL18BP105 (SEQ ID NO:15) immunity more effective (table 7).

These results show poxvirus subunit peptide, vIL18BP105 (SEQ ID NO:15), with anti--induce cell and the humoral immune reaction in the HLA-DR04tg mice during HLA-DR antibody L243 coupling.The T-cell proliferative response of indicator cells mediation immunity especially strengthens because of antigen-L243 conjugate.In the CIL18BP105 immune mouse, antibody produces quickly and rises.Peptide-antibody coupling matter specific ionization peptide is earlier induced higher antibody titer.These results show can be through coming successfully to cause with the L243 coupling to the antigenic T-cell effect of relatively little peptide.

The interferon gamma of table 7. immune mouse produces

Conclusion

Need Th1 and Th2 immunoreation, cell-mediated and humoral immunization to the vaccine of poxvirus, and suitable memory cd4 t cell pond (Belyakoc etc., Proc.Natl.Acad.Sci.USA 100:9458-9463,2003).The result who is provided shows with the link coupled subunit antigen of APC-targeting antibodies can strengthen and induce Th1, Th2, and humoral immune reaction.

The nasal administration of embodiment 3. subunit vaccines

Vaccine (15-25 μ g peptide altogether) (10 microlitres/nostril) is used in mice (HLA-DR04Tg) anesthesia and intranasal (i.n).Vaccine is a free peptide, or peptide-L243 conjugate, and in these experiments, adjuvant is the calcium phosphate adjuvant of being described by (Clin Diagnos Lab Immunol 9:1021-1024,2002) such as He (10 μ g/ doses of antigen).Contrast is by immunity (originally) mice, through intact virus albumen (i.n.) mice immunized, general immunity mice (peptide, subcutaneous (s.c)) with only form through carrier/adjuvant (i.n) mice immunized.Use the peptide of equivalent in each case.2 weeks (d14) and 4 weeks (d28) after swashing in advance, use with preparatory approach identical when sharp with twice of mice booster immunization.Can use the combination (for example, s.c when swashing in advance, then i.n. immunity in the 14th day) of immunization route.

Swashed immunity in advance the 35th day afterwards, five mices in each treatment group are put to death (25/75 mice).Before in advance swashing immunity (d0), the 7th day, the 28th day and the 56th day, results serum.In addition, results nasal cavity lavation (NL) liquid or bronchoalveolar lavage (BAL) liquid and splenocyte when putting to death.

Antigen-specific antibodies in respiratory tract fluid (when putting to death, collecting through NL or BAL) and the serum comes titration through serial dilution and together with contrasting from all treatment groups fixing complete recombinant antigen of (comprising mice originally) (or cowpox albumen), peptide, irrelevant peptide and serial dilution serum is applied to ELISA.Measure the homotype of specific antibody.

Neutralizing antibody is present in through nasal cavity or subcutaneous administration to come in the mice immunized.Antibody and antigenic peptides and the reaction of whole virus protein.Nasal administration can more effectively promote mucosal immunoreaction, and subcutaneous administration can more effectively promote the systemic immune response to poxvirus.

Embodiment 4. is through butt joint-prepare the alternative method of immune conjugate with-locking (DNL) technology

DDD and AD fusion rotein

The DNL technology can be used for making the dimer that comprises almost any antibody or its fragment or other protein or peptide moiety, trimer, the tetramer, six aggressiveness etc.For certain preferred embodiments, can produce the IgG antibody, the F (ab ') that are the fusion rotein form that contains dimerization and butt joint territory (DDD) or grappling territory (AD) sequence ₂Antibody fragment and subunit antigen peptide.Though in preferred embodiments, produce the DDD and the AD part that are the fusion rotein form, those skilled in the art will recognize that other coupling method in opinion method and composition scope capable of using, like chemical crosslinking.

The DNL construct can through will for example resist-the Fab-DDD fusion rotein of HLA-DR or anti--CD74 antibody and vIL18BP105-AD fusion rotein make up and form.Perhaps, can produce construct with the combination of IgG-AD fusion rotein and vIL18BP105-DDD fusion rotein.This technology be do not have restrictive and can produce any useful proteins matter of being AD or DDD fusion rotein form or peptide so that incorporate in the DNL construct.When utilizing chemical crosslinking, AD and DDD conjugate are not limited to protein or peptide and can comprise any molecule that can use any crosslinking technological known in the art and AD or DDD sequence crosslinked.

Can be each DDD or the AD fusion rotein is developed independently transgenic cell line.In case produce, but module purification (if desired), or be maintained in the cell culture supernatant.After the generation, any DDD fusion rotein module can with any AD fusion rotein module combinations to produce the DNL construct.For dissimilar constructs, different AD capable of using or DDD sequence.Exemplary DDD and AD sequence are provided below.

DDD1：

SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：24)

DDD2：

CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：25)

AD1：QIEYLAKQIVDNAIQQA(SEQ?ID?NO：26)

AD2：CGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO：27)

Expression vector

Plasmid vector pdHL2 has been used to produce many antibody and based on the construct of antibody.Referring to Gillies etc., J Immunol Methods (1989), 125:191-202; Losman etc., Cancer (Phila) (1997), 80:2660-6.Synthesizing of the heavy chain of bicistronic mRNA mammalian expression vector guiding IgG and light chain.For many different I gG-pdHL2 constructs, the carrier sequence is that identical, unique difference is variable domain (VH and VL) sequence mostly.Use biology tool well known by persons skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab-AD expression vector.In order to produce the Fab-DDD expression vector, the coded sequence in the CH2 of hinge, heavy chain and CH3 territory is replaced with the sequence of preceding 44 residues of preceding 4 residues, 14 residue Gly-Ser junctional complexs and the people RII α (being called as DDD1) of coding hinge.In order to produce the Fab-AD expression vector; The sequence in hinge, CH2 and the CH3 territory of IgG is replaced with preceding 4 residues, the 15 residue Gly-Ser junctional complexs of coding hinge and is called the sequence of the synthetic AD (being called as AD1) of 17 residues of AKAP-IS, and it uses bioinformatics and peptide array technique to produce and shows the dimer with very high affinity (0.4nM) combination RII α.Referring to Alto, wait Proc.Natl.Acad.Sci., U.S.A (2003), 100:4445-50.

Two kinds of shuttle vectors are converted into Fab-DDD1 or Fab-AD1 expression vector with promotion with the IgG-pdHL2 carrier through design.Use this technology, we have produced and have been used for multiple known antibodies, AD and/or the DDD fusion rotein and the coding plasmid of expressing like the Fab of hLL1, hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many other antibody.

Obtain trimer DNL construct through the AD fusion rotein that makes the DDD fusion rotein that comprises IgG antibody for example or F (ab) antibody fragment and comprise subunit antigen peptide for example with molar ratio reaction between 1.4: 1 and 2: 1.In the PBS that contains 1mM EDTA, total protein concentration is 1.5mg/ml.Subsequent step can relate to TCEP reduction, HIC chromatography, DMSO oxidation and affinity chromatography to obtain purification DNL construct.Add 5mM TCEP and cause forming a apace ₂The b complex.Show that in conjunction with test antibody moiety and antigenic peptides part keep its antigen respectively and combine and antigenic functional character.

Use this technology, can be through preparing each self-contained complementary DDD and AD suitable fusion rotein partly with almost any antibody or antibody fragment are connected to any subunit antigen peptide.

Following peptide is processed and is useful on the AD2 module of the catenation sequence that connects the subunit vaccine peptide.AD2 peptide fusant is partly made up to provide and to have the vaccine based on subunit of APC targeting antibodies or antibody fragment with IgG that is connected DDD2 or Fab.

ND8L

SIFLQVLDHKNVYFQGGGSCGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO：34)

CD8L

CGQIEYLAKQIVDNAIQQAGCGGGSSIFLQVLDHKNVYFQ(SEQ?ID?NO：35)

NIL18BP

HYRFTCVLTTLNGVSGGGSCGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO：36)

CIL18BP

CGQIEYLAKQIVDNAIQQAGCGGGSHYRFTCVLTTLNGVS(SEQ?ID?NO：37)

CSCRD8L

CGQIEYLAKQIVDNAIQQAGCGGGSYHQFVIDQLKLSVNF(SEQ?ID?NO：38)

CSCRIL18BP105

CGQIEYLAKQIVDNAIQQAGCGGGSGNCTFVTYLRHLSTV(SEQ?ID?NO：39)

Embodiment 5. is used for the Liposomal formulation of nasal administration based on the vaccine of subunit

The Liposomal formulation for preparing antigenic peptides and L243 antibody coupling through standard technique.The intranasal peptide has junctional complex through design at C-end and N-end.The terminal junctional complex of C-is used for coupling L243 antibody.The terminal junctional complex of N-is used for promoting to be connected with liposome via palmitoylation.Peptide conjugate is as follows.The CD8L118 peptide is not lipoprotein and is encapsulated in the liposome.

L1R183

GVQFYMIVIGVIILAALF(SEQ?ID?NO：40)

Link coupled L1R183

KKKKGVQFYMIVIGVIILAALFPSEC(SEQ?ID?NO：41)

Link coupled A27L3

KSGTLFPGDDDLAIPATEFFSTKAAKKPSEC(SEQ?ID?NO：42)

Link coupled IL18BP105

KSHYRFTCVLTTLNGVSPESC(SEQ?ID?NO：43)

CD8L118

HDDGLIIISIFLQVLDHKNVYFQKIGGGSC(SEQ?ID?NO：44)

Fig. 9 (A) shows the result of the subunit vaccine of nasal administration liposome formulation.Prepare peptide and and antibody coupling like above embodiment 1 and 2 descriptions.The result who provides among Fig. 9 be presented to after the mice nasal cavity immunity in response at external T cell proliferation of hatching with specified polypeptide.Observe the strongest effect (Fig. 9 A) to the T cell proliferation for deriving from the antigenic L1R183 antigenic peptides of L1R (SEQ ID NO:39), said L1R antigen provides interior mature virion (IMV) albumen of immundominance cell of post-exposure prophylaxis.The independent naked peptide of showing with liposome carries out immunity (Fig. 9 B) to mice the T cell proliferation is produced few influence, no matter test peptides why.With not existing free peptide or empty liposome immunity under the liposome situation also the T cell proliferation almost not to be had influence (data are not shown).The result shows that the coupling of using with the APC targeting antibodies comes the poxvirus vaccine effectively induction of immunity reaction of nasal administration based on subunit.

***

In view of the disclosure, need not too much test and to produce and to use all compositionss and method disclosed herein and opinion.Though describe these compositionss and method according to preferred embodiment; But those skilled in the art show and are prone to know that the step or the sequence of steps of compositions and method and methods described herein can adopt version, and do not deviate from notion of the present invention, spirit and scope.More particularly, the medicament instead medicament as herein described that some chemistry is relevant with physiology will obtain same or similar result simultaneously.Those skilled in the art show and are prone to all these type of similar replacements of knowledge and revise all be regarded as in defined the present invention's spirit, scope and notion like the accessory claim book.

Claims (26)

1. immune conjugate, it comprises:

A) at least a from the proteic antigenic peptides of poxvirus; With

B) conjugated antigen is antibody or its Fab of delivery cell (APC), wherein said antibody or fragment and said antigenic peptides coupling;

Wherein said immune conjugate is used to the experimenter and induced immunoreation to said poxvirus.

2. immune conjugate according to claim 1, wherein said poxvirus albumen is the virus immunity regulatory factor.

3. immune conjugate according to claim 2, wherein said poxvirus albumen are viral IL-18 conjugated protein (vIL18BP).

4. immune conjugate according to claim 1, wherein said poxvirus albumen is envelope protein.

5. immune conjugate according to claim 1, wherein said poxvirus albumen is selected from the group of being made up of L1R, A27L and D8L.

6. immune conjugate according to claim 1, wherein said immune conjugate comprise at least a antigenic peptides and at least a antigenic peptides from virus envelope protein from the virus immunity regulatory factor.

7. immune conjugate according to claim 1, wherein said poxvirus is a variola.

8. immune conjugate according to claim 1, wherein said antibody or its fragment combine to be selected from the APC antigen by the following group of forming: HLA-DR, CD74, CD209 (DC-SIGN), CD34, CD74, CD205, TLR2 (toll appearance receptor 2), TLR4, TLR7, TLR9, BDCA-2, BDCA-3 and BDCA-4.

9. immune conjugate according to claim 1, wherein said APC antigen is HLA-DR or CD74.

10. immune conjugate according to claim 9; Wherein said antibody or its fragment are anti--HLA-DR antibody, and it comprises heavy chain complementary determining region (CDR) sequence C DR1NYGMN (SEQ ID NO:1), CDR2 WINTYTREPTYADDFKG (SEQ ID NO:2) and CDR3 DITAVVPTGFDY (SEQ ID NO:3) and light chain CDR sequence C DR1 RASENIYSNLA (SEQ ID NO:4), CDR2 AASNLAD (SEQ ID NO:5) and CDR3OHFWTTPWA (SEQ ID NO:6).

11. immune conjugate according to claim 9; Wherein said antibody or its fragment are anti--CD74 antibody, and it comprises light chain CDR sequence C DR1RSSQSLVHRNGNTYLH (SEQ ID NO:7), CDR2 TVSNRFS (SEQ ID NO:8) and CDR3 SQSSHVPPT (SEQ ID NO:9) and heavy chain CDR sequence C DR1NYGVN (SEQ ID NO:10), CDR2 WINPNTGEPTFDDDFKG (SEQ ID NO:11) and CDR3 SRGKNEAWFAY (SEQ ID NO:12).

12. immune conjugate according to claim 1, wherein said antigenic peptides has the aminoacid sequence that is selected from by the following group of forming: SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

13. immune conjugate according to claim 1, wherein said immune conjugate are the fusion rotein that comprises said antigenic peptides and said antibody or antibody fragment.

14. immune conjugate according to claim 1, wherein said antigenic peptides and said antibody or antibody fragment are covalently bound.

15. immune conjugate according to claim 1, wherein said antigenic peptides are the parts of first fusion rotein, said antibody or its fragment are the parts of second fusion rotein, and said first and second fusion rotein are bonded to each other.

16. immune conjugate according to claim 15; Wherein first fusion rotein comprises dimerization and butt joint territory (DDD) part from human protein kinase A (PKA) RI α, RII α, RI β or RII β, and said second fusion rotein comprises from the proteic grappling of AKAP territory.

17. a pharmaceutical composition, it comprises immune conjugate according to claim 1 and pharmaceutically acceptable carrier.

18. pharmaceutical composition according to claim 17, wherein said pharmaceutical composition is a subunit vaccine.

19. pharmaceutical composition according to claim 18 wherein provides the immunity to poxvirus infection with said vaccine administration to the experimenter.

20. pharmaceutical composition according to claim 18 is wherein induced the immunity that infects to variola with said vaccine administration to the experimenter.

21. pharmaceutical composition according to claim 18, wherein said compositions further comprises at least a adjuvant.

22. induce the method to the immunity of poxvirus infection for one kind, it comprises to the experimenter uses subunit vaccine according to claim 18.

23. method according to claim 22, wherein said poxvirus is a variola.

24. method according to claim 22, the subcutaneous or nasal administration of wherein said compositions.

25. method according to claim 24, wherein liposome subunit vaccine via intranasal application is used.

26. method according to claim 17, wherein said immune conjugate are with the expression vector administered of encoding fusion protein, said fusion rotein comprises at least a from the proteic antigenic peptides of poxvirus and the antibody or its Fab that combine APC.

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