CN102747103A - AIDS vaccine of double-deletion replicative vaccinia virus based on A52L gene and B8R gene - Google Patents
AIDS vaccine of double-deletion replicative vaccinia virus based on A52L gene and B8R gene Download PDFInfo
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Abstract
The invention relates to relates to an AIDS vaccine of double-deletion replicative vaccinia virus based on A52L gene and B8R gene, which comprises the replicative vaccinia virus capable of expressing human immunodeficiency virus (HIV) antigen. The invention also relates to a method for constructing the replicative vaccinia virus, and an immunization inoculation scheme capable of using the AIDS vaccine containing the replicative vaccinia virus.
Description
Technical field
The present invention relates to the antiviral immunity field.More specifically, the present invention relates to a kind of rf attenuated vaccinia virus carrier, based on the AIDS vaccine of said carrier, and the preparation method and the purposes of said vaccine.
Background technology
(Acquired Immunodeficiency Syndrome AIDS) is called for short AIDS to AIDS, is that (Human Immunodeficiency Virus HIV) infects a kind of communicable disease that causes by the human immunodeficiency virus.Since 1981 found the first routine case, AIDS spread in the whole world always with surprising rapidity, became one of the most serious virus disease of harm humans life and health in the world.
Global popular high pathogenicity rate and the case fatality rate of having caused of HIV is the infection of control HIV, and traditional chemotherapy, immunotherapy and new method are able to continuous research, development.Most HIV the infecteds are carried out high reactivity antiviral therapy (HAART); Though can effectively suppress viremia; Delay the course of disease; But can not remove the virus (Tussey LG, the et al.:Antigen burden is amajor determinant of human immunodeficiency virus-specific CD8_T cellmaturation state:Potential implications for therapeutic immunization.J InfectDis 2003 that hide; 187:364-374.).
Protectiveness vaccine inoculation is to solve control HIV to infect the optimal path of global problem.For this reason, scientists has been carried out the research of attenuated live vaccine, inactivated vaccine, recombinant subunit vaccine, dna vaccination and various live vector vaccine (being the vaccine of carrier with adenovirus, canary pox virus, influenza virus, vaccinia virus, rabies virus, herpes stomatitis virus, Corynebacterium diphtheriae, suis etc. for example).Though attenuated live vaccine is proved in simian immunodeficiency virus (SIV) infection model effectively; But, initial immunity has certain lethality rate (Baba TW in growing up macaque; Jeong YS; Penninck D, et al.1995.Pathogenicity of live, attenuated SIV after mucosal infection of neonatalmacaques.Science 267:1820-25).Because the risk of rf attenuated live vaccine and evidence suggests that it does not have extensive protectiveness, the application of attenuated live vaccine waits further research.Research shows that in the orangutan model, inactivated vaccine can not protect HIV to attack, can not inducing cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) reaction.Live vector vaccine and dna vaccination all not only can the inducing cell immunoreations but also can have been induced humoral immune reaction, and the combined immunization of expressing antigenic live vector vaccine of HIV and dna vaccination becomes the new vaccine research strategy of control HIV infection.
In the research of live vector vaccine, (for example, vaccinia virus Tiantan strain (vaccinevirus TianTan strain, VTT)) carries out the most deeply with extensive for the live vector vaccine research of carrier with vaccinia virus.That the vaccinia virus recombinant vaccine has is effective, convenient to Heat stability is good, inoculation, do not need advantage such as adjuvant; Yet produce heavier local reaction behind the vaccinia virus recombinant vaccine inoculation human body, particularly in a certain proportion of immunodeficient patient, can cause severe complications.Express some immunogenic vaccinia virus recombinant vaccine and fail to reach the ideal immune effect, the security that how can further improve vaccinia virus vector simultaneously in the immunogenicity that strengthens the vaccinia virus recombinant vaccine, scholars have made a lot of deep researchs.Along with new virus disease constantly occurs; And understanding in depth to the host anti-virus immune mechanism; The multiple antigen of the same cause of disease of construction expression, the multiple antigenic polyvalent recombinant vaccinia virus vector of Different Kinds of Pathogens, and it is significant in the vaccinia virus recombinant carrier, to seek more multistable fixed effective foreign gene insertion district.
Summary of the invention
In one aspect, the present invention provides a kind of replicative vaccinia virus, and the A52L gene and the B8R gene of wherein said vaccinia virus gene group lack simultaneously, and has inserted the antigenic polynucleotide of at least a coding HIV in the TK district.
Of the present invention one preferred embodiment in, replicative vaccinia virus is a vaccinia virus Tiantan strain.
In an embodiment of the present invention, the antigenic polynucleotide of said at least a coding HIV derive from HIV-1B '/C reorganization hypotype 97CN54 strain.In some concrete embodiments, said HIV antigen is selected from one or more among env, gag and the pol.In a concrete embodiment, env, gag and pol are inserted replicative vaccinia virus of the present invention simultaneously.
Another aspect of the present invention provides replicative vaccinia virus of the present invention to be used for preventing or treating the purposes of the vaccine composition of individual HIV virus infection in preparation.
Another aspect of the present invention provides a kind of AIDS vaccine, and it comprises the antigenic replicative vaccinia virus of expression HIV of the present invention.
The present invention also provides the immunization scheme of using vaccine of the present invention.
Preservation information
Replicative vaccinia virus (Vaccinia virus) VTT Δ B8R is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), preserving number CGMCC 4714 on March 30th, 2011.
Replicative vaccinia virus (Vaccinia virus) VTT Δ A52L/B8R-GPE is preserved in CGMCC, preserving number CGMCC 4713 on March 30th, 2011.
The ETEC (Escherichia coli) that contains transferring plasmid pSKA52LLacZ is preserved in CGMCC, preserving number CGMCC 4722 on March 30th, 2011.
The ETEC (Escherichia coli) that contains transferring plasmid pSKA52L-GNA is preserved in CGMCC, preserving number CGMCC 4723 on March 30th, 2011.
Description of drawings
Fig. 1: show transferring plasmid pSKA52LlacZ design of graphics.
Fig. 2: show transferring plasmid pskA52LR-GNA design of graphics.
Fig. 3: show that pcr amplification detects the result of the recombinant virus VTT Δ A52LLacZ of A52L genetically deficient.Swimming lane M is dna molecular amount mark DL15000 (Takara company); Swimming lane 2 is an A52L deletion mutantion district amplified fragments.
Fig. 4: the PCR Screening and Identification result who shows recombinant virus VTT Δ B8R/A52L.Swimming lane 1-4 shows the purification of Recombinant virus that the GFP gene has been deleted; Swimming lane 5,23 shows the maternal viral VTT Δ B8R that reorganization does not take place; Swimming lane 6-22 shows the not purification of Recombinant virus that contains the GFP gene; Swimming lane 24 is dna molecular amount mark DL2000 (Takara company).
Fig. 5: the enzyme that shows plasmid pSC65gagpol-1455M is cut qualification result.Swimming lane 1:DNA molecular weight marker DL15000 (Takara company); Swimming lane 2:XmaI/PacI double digestion plasmid pSC65gagpol-1455M; Swimming lane 3:PacI single endonuclease digestion plasmid pSC65gagpol-1455M; Swimming lane 4; Plasmid Psc65gagpol-1455M.
Fig. 6: the PCR qualification result that shows vaccinia virus recombinant VTT Δ A52L/B8R-GPE.Swimming lane 1-3 is recombinant virus VTT Δ A52L/B8R-GPE DNA; Swimming lane 4 is recombinant virus VTT Δ B8R/A52L DNA; Swimming lane 5 is dna molecular amount mark DL15000 (Takara company).
Fig. 7: the western blot qualification result that shows vaccinia virus recombinant VTT Δ A52L/B8R- GPE.Swimming lane 1 and 4 be preparatory dsred protein molecular weight marker (Precision Plus Protein Dual Color Standards, Bio-Rad).Swimming lane 2-3 is the gp1455M Detection of antigen result of VTT Δ A52L/B8R-GPE, and swimming lane 5-6 is the gag/pol Detection of antigen result of VTT Δ A52L/B8R-GPE.
Fig. 8: show viral VTT, VTT Δ B8R, VTT Δ A52LlacZ and VTT Δ B8R/A52L growth curve mensuration result in CEF, Vero, Hela cell respectively.
Fig. 9 A-9B: the interior virulence experimental result of rabbit that shows recombinant virus.Virus VTT, VTT Δ A52LLacZ and VTT Δ A52L/B8R-GPE are respectively with 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4Pfu/100 μ L is injected in the NZw back intracutaneous of shaving the light dorsal body setae.The continuous 14 days red and swollen situation in observed and recorded injection of skin position, and with slide calliper rule survey its infiltration diameter and downright bad diameter.A shows that 1-14 days each extent of dilution skin decrease the diameter summation; B shows the 7th day, and each extent of dilution skin decreases the diameter summation.
Figure 10: the mouse collunarium virulence experiment that shows recombinant virus.
Figure 11 A-Figure 11 C: show that respectively Elispot detects the result that DNA initial immunity/recombinant vaccinia booster immunization strategy mouse anti-HIV env T cell produces IFN-γ, IL-2 and IL-4.
Figure 12: show that Elispot detects the result that continuous two pin recombinant vaccinia immunization strategy mouse anti-HIV env T cells produce IFN-γ.
Figure 13 A-Figure 13 B: show phase mouse cell and humoral immunity level when the continuous two pin recombinant vaccinia immunization strategies of detection are long respectively.A shows that Elispot detects the result that mouse anti-HIV env T cell produces IFN-γ; B shows that the ELISA method detects the result of Env specific IgG antibodies level.
Embodiment
(Hmuan Immunodeficiency Virus HIV) is a kind of retrovirus to the human immunodeficiency virus, and it mainly attacks and destroy the CD4 that activate immunity reacts after getting into blood of human body
+The T lymphocyte makes it lose original normal immunologic function.HIV infects and causes AIDS (abbreviation AIDS) the mankind.Protectiveness vaccine inoculation is to solve control HIV to infect the optimal path of global problem.
Vaccine to AIDS of the present invention is based on replicative vaccinia virus and makes up, its be different from present conventional use like non-replicating vaccinia virus vectors such as MVA (modified vaccinia Ankara) strain, NYVAC (New YorkVaccinia virus) strain and ALVAC (canarypox virus) strains.Therefore, in one aspect, the present invention provides a kind of replicative vaccinia virus, and the A52L gene and the B8R gene of wherein said vaccinia virus gene group lack simultaneously, and has inserted the antigenic polynucleotide of at least a coding HIV in the TK district.
Term " replicative vaccinia virus " is meant the vaccinia virus vector that can in human body, duplicate.
Of the present invention one preferred embodiment in, said replicative vaccinia virus be vaccinia virus Tiantan strain (vaccine virus TianTan strain, VTT).After last century, the '20s separated, the Ceng Zuowei antismallpox vaccine extensively inoculated in China vaccinia virus Tiantan strain, and has finally eliminated smallpox in China by the Chinese science man.Tiantan strain vector can efficiently express foreign protein, and immunogenicity is good, can induce stronger humoral immunization and cellular immunization in vivo, and the immunoreactive time length also is longer than nonreplication vector.
In replicative vaccinia virus of the present invention, the A52L gene and the B8R gene of vaccinia virus gene group lack simultaneously.
" B8R gene " extensively is present in the vaccinia virus; The homologous protein of its coding solubility IFN-γ acceptor can competitively combine the IFN-gamma receptor gene, in BA (the Paulo H.Verardi of IFN-γ aspect antiviral and immunomodulatory; Leslie A.Jones; Fatema H.Aziz, Shabbir Ahmad, and Tilahun D.Yilma*.Vaccinia Virus Vectors with an Inactivated GammaInterferon Receptor Homolog Gene (B8R) Are Attenuated In Vivo without aConcomitant Reduction in Immunogenicity.Journal of Virology; January 2001; Vol.75, No.1, p.11-18).
" A52L gene " is present in the conservative gene that comprises in 16 strain vaccinia virus that the Temple of Heaven strain etc. identified, and its expression product can interact with one group of chemokine, stop chemokine to form concentration gradient, the immunoreation that virus is resisted body by this.The A52L gene is at American-European main flow bovine vaccine strain called after A41 (Richard H.Clark; Julia C.Kenyon; Nathan W.Bartlett; David C.Tscharke and Geoffrey L.Smith.Deletion of gene A41L enhances vaccinia virusimmunogenicity and vaccine efficacy.Journal of General Virology (2006), 87,29-38DOI 10.1099; Bahar MW; Kenyon JC, Putz MM, Abrescia NGA; PeaseJE; Et al. (2008) Structure and function of A41, a vaccinia virus chemokinebinding protein.PLoS Pathog 4 (1): e5.doi:10.1371/journal.ppat.0040005), at the Temple of Heaven strain called after TA52L.
The inventor is surprised to find, and vaccinia virus Tiantan strain its virulence when exciting significant immunne response that lacks B8R and A52L gene simultaneously but significantly reduces.This discovery makes can make up the AIDS vaccine with greater security.
Term " TK district " refers to the encoding sox section of the thymidine kinase of vaccinia virus gene group, and the expression of vaccinia virus normal function does not need this section, after it is replaced by foreign DNA, can not influence virus genomic replication.In AIDS vaccine of the present invention, as the genomic TK district of the replicative vaccinia virus of carrier having inserted the antigenic polynucleotide of at least a coding HIV.
Can select appropriate H IV antigen gene to be used to make up vaccine of the present invention, for example, can select the antigen gene of local main epidemic strain to be used for vaccine construction according to the epidemiological study result.In an embodiment of the present invention; The antigenic polynucleotide of said at least a coding HIV that insert replicative vaccinia virus TK of the present invention district derive from HIV-1B '/C reorganization hypotype 97CN54 strain (Su L; Graf M; Zhang Y; Von Briesen H, Xing H,
J; Melzl H; Wolf H, Shao Y, Wagner R..Characterization of a virtually full-length humanimmunodeficiency virus type 1 genome of a prevalent intersubtype (C/B ') recombinant strain in China.J Virol.2000Dec; 74 (23): 11367-76.).HIV-1B '/C reorganization hypotype 97CN54 strain is the main epidemic isolates of HIV of China, and this strain for example can be available from CDC aids prevention control center.In an embodiment of vaccine of the present invention; Thymidine kinase (TK) district at replicative vaccinia virus has inserted the antigenic polynucleotide of at least a coding HIV; Preferably, the polynucleotide that inserted are polynucleotide of coding HIV-1 antigen env, gag and pol.
Can adopt multiple suitable means to make up replicative vaccinia virus of the present invention.For example, in an embodiment of the present invention, adopt general transfer vector pSKA52LLacZ of the present invention, foreign gene (LacZ) is recombinated in the A52L district of vaccinia virus gene group DNA, and delete the A52L gene simultaneously.The general transfer vector pSKA52LLacZ of vaccinia virus of the present invention is the transferring plasmid that contains LacZ genescreen mark.This transferring plasmid vector contains following element: 1. two selection markers: Amp resistant gene, LacZ gene.Be used for the blue hickie screening of vaccinia virus recombinant by the LacZ gene of p7.5 promotor startup.2. homology arm A52LL and A52LR sequence: A52LL and A52LR are the part fragments of vaccinia virus A52L gene, are the homologous sequences that intermolecular homologous recombination takes place for vaccinia virus and transferring plasmid.3. the promotor pE/L in late period morning of vaccinia virus.4. MCS is positioned at the downstream of promotor pE/L.
In a concrete embodiment, the inventor has made up attenuated carrier VTT Δ B8R/A52L (for B8R and A52L Gene Double disappearance, not with the bovine vaccine the Temple of Heaven strain of LacZ selective marker).On this basis, further insert HIV B '/C type CN54 strain HIV-1env, gag and pol gene, obtained replicative vaccinia virus VTT Δ A52L/B8R-GPE in the TK district.
Another aspect of the present invention provides replicative vaccinia virus of the present invention to be used for preventing or treating the purposes of the vaccine composition of individual HIV virus infection in preparation.
Another aspect of the present invention provides a kind of expression HIV antigenic AIDS vaccine, and it comprises replicative vaccinia virus of the present invention.
The present invention also provides the immunization scheme of using vaccine of the present invention.
According to an aspect of the present invention, immunization scheme employing of the present invention " two pin immunization strategies ".Term " two pin immunization strategies " means twice or the repeatedly immunity that is different from different vaccine forms, and vaccine form of the same race is all used in twice (initial sum reinforcement) immunity before and after being meant, for example adopts the vaccinia virus that makes up according to the present invention.
The inventor finds, uses with the vaccinia virus that makes up according to the present invention to 4 all " two pin immunization strategies " at interval between twice, twice of the AIDS vaccine sequential immunization mouse of carrier, can excite significant immunne response.
According to another aspect of the present invention; Immunization scheme of the present invention adopts " Prime-boost strategy ", and promptly (Liu Ying etc. express the structure and the Immunogenicity Study of the vaccinia virus recombinant of HIV polyvalent antigen with the initial immunity of HIV dna vaccination earlier; Virus journal 2003; Vol 19 (3): 205-209), carry out the immunization protocol of booster immunization again with replicative vaccinia virus of the present invention, can successfully induce body to produce high-caliber body fluid and cell immune response.
The genetic modification that replicative vaccinia virus vector of the present invention relates to does not change its replicability; And can significantly reduce the virulence of carrier; Improve security, can be used for the polyvalent vaccine of a plurality of antigens of the same cause of disease of construction expression (for example, the multiple not synantigen of HIV).AIDS vaccine based on replicative vaccinia virus of the present invention can be induced high-caliber anti-HIV body fluid and cellullar immunologic response, and the security that effectively improves the replicative vaccinia virus AIDS vaccine.
Replicative vaccinia virus vector of the present invention can also be used for the recombiant vaccine of the multiple antigen (for example, antigens such as cause of disease not of the same race or tumour) of a plurality of cause of diseases of construction expression.
Below in conjunction with specific embodiment the present invention is done further elaboration.
Embodiment 1: transferring plasmid
1, transferring plasmid pSKA52LLacZ
Transferring plasmid pSKA52LLacZ is made up and preservation (preserving number: CGMCC4722) by the inventor.This plasmid is used for deleting the A52L gene of vaccinia virus Tiantan strain genomic dna, and inserts the LacZ gene in this zone simultaneously.The left and right sides homology arm A52LL and the A52LR that comprise vaccinia virus Tiantan strain A52L gene on the plasmid are inserted with the LacZ gene under the control of vaccinia virus promotor in the middle of the homology arm of the left and right sides.Plasmid construction figure sees Fig. 1.
2, transferring plasmid pSKA52L-GNA
Transferring plasmid pSKA52L-GNA is made up and preservation (preserving number: CGMCC4723) by the inventor.This plasmid is used for deleting the A52L gene of vaccinia virus Tiantan strain genomic dna; The left and right sides homology arm A52LL and the A52LR that comprise vaccinia virus Tiantan strain A52L gene on the plasmid, the direct repeat sequence of the A52L gene about middle GFP gene, neo gene and the 200bp that is inserted with under the control of vaccinia virus promotor of left and right sides homology arm.Plasmid construction figure sees Fig. 2.
Embodiment 2: based on the structure of the replicative vaccinia virus vector of A52L genetic modification
1, the structure of A52L single-gene disappearance replicative vaccinia virus vector VTT Δ A52LlacZ
Infect 80-90% CEO in blocks for inoblast (MOI=0.01～0.1) with vaccinia virus Tiantan strain VTT (available from the Beijing Tiantan Biological Products Co.ltd); 37 ℃ of absorption are after 1 hour; Supernatant discarded, and use serum-free Eagle ' s substratum rinsing cell 2 times.According to Invitrogen company transfection reagent box (Lipofectin Reagent Cat.18292-011) specification sheets, above-mentioned through the CEF of virus infection cell with transferring plasmid pSKA52LLacZ transfection.Cultivate after 48 hours, harvested cell and supernatant are stored in-20 ℃ after the freeze thawing three times.Get 10 μ l transfection liquid and inoculate 80% CEF cell in blocks, after 48 hours with nutrient agar medium (Eagle ' s that contains 10mg/mL X-gal, 1% toluylene red, 1% low melting-point agarose) shop spot, the isolated locus coeruleus virus of picking at random, 5 generations of continuous single spot purifying.Extract recombinant virus dna and adopt PCR method to identify, use following primer:
Forward primer: 5 '-CAATCTAgAggAggAAggACgTAATgCATg-3 ' (SEQ ID NO.:1)
Reverse primer: 5 '-TACggTACCTCATCCATTAgAgAgTTAg-3 ' (SEQ ID NO.:2)
The result shows that vaccinia virus recombinant amplifies the A52L district specific fragment of 4560bp, confirms that the A52L district of vaccinia virus recombinant has inserted the LacZ gene and successfully lacked the A52L gene simultaneously.Vaccinia virus recombinant called after VTT Δ A52LLacZ.The PCR qualification result is as shown in Figure 3.
2, the structure of A52L/B8R Gene Double disappearance replicative vaccinia virus vector
2.1, transferring plasmid pSKA52L-GNA transfection
Tiantan strain vaccinia virus VTT Δ B8R (preserving number: CGMCC 4714) with B8R genetically deficient infects 80-90% CEO in blocks for inoblast (CEF) (MOI=0.01～0.1); 37 ℃ of absorption are after 1 hour; Discard culture supernatant, and use serum-free Eagle ' s substratum rinsing cell 2 times.According to Invitrogen company transfection reagent box (Lipofectin Reagent 2000Cat.18292-011) specification sheets, above-mentioned through the CEF of virus infection cell with transferring plasmid pSKA52L-GNA transfection.37 ℃ of CO
2Incubator is cultivated 24h, and harvested cell and supernatant are stored in-20 ℃ after the freeze thawing three times.
2.2, screening and the evaluation of recombinant virus VTT Δ B8R/A52L
2.2.1, CEF cell in blocks cultivates 24h with Eagle ' the s substratum that contains 0.5mg/ml G418 (available from GIBCO company) earlier in advance; 10 times of serial dilutions of transfection virus liquid of results are inoculated into pretreated CEF cell, and nutrient solution is removed in the 1-2h hypsokinesis, adds to contain 1% low melting-point agarose (available from Promega company); 0.5mg/ml the Eagle ' s of G418 (available from GIBCO company); Cultivate 48h for 37 ℃, fluorescent microscope is observed down, the positive plaque of picking GPF.Plaque multigelation 3 times is got 10 times of serial dilutions of 100ul again, is inoculated into the pretreated CEF cell of G418.Repeat said process, single spot continuous purification 2-3 generation.
2.2.2, with after the positive plaque freeze thawing of GPF three times; Get 10 times of serial dilutions of 100ul, the CEF cell that inoculation is handled without G418, nutrient solution is removed in the 1-2h hypsokinesis; Add the Eagle ' s that contains 1% low melting-point agarose (available from Promega company); Cultivate 48h for 37 ℃, fluorescence microscope, the negative plaque of picking GPF.After the plaque freeze thawing three times, get 100 μ l and insert 24 orifice plates, behind the cultivation 48h, extract DNA, PCR identifies, uses following primer:
Forward primer: 5 '-CAATCTAGAGGAGGAAGGACGTAATGCATG-3 ' (SEQ ID NO.3)
Reverse primer: 5 '-TACGGTACCTCATCCATTAGAGAGTTAG-3 ' (SEQ ID NO.4)
The plaque that PCR result is correct gets into the next round purifying.Repeat said process, until the complete purifying of plaque.The PCR qualification result is seen Fig. 4.
Embodiment 3: based on the structure of the replicative vaccinia virus vector AIDS vaccine of A52L genetic modification
1, the structure of transferring plasmid pSC65gagpol-1455M
PSC65 plasmid (CGMCC:1097) to contain vaccinia virus TK gene left and right sides homology arm is the basis, between its left and right arms, embeds the gagpol gene of HIV-1B '/C recombinant strain CN54 and the env gene 1455M that codon is optimized.The gagpol gene is directly from pVTT-gagpolenv plasmid (CN101020052; One Chinese patent application 200610007567.7) goes up to get off with PmeI/SmaI (available from NEB company) double digestion; Behind sticking end-filling; Be connected into also to mend in the flat pSC65 plasmid behind HindIII (available from the NEB company) single endonuclease digestion with the T4DNA ligase enzyme, be built into recombinant plasmid pSC65gagpol.
With plasmid pDRVISV1455M (CGMCC No.2514) is masterplate, and the 1455M gene that the pcr amplification codon is optimized wherein uses following primer:
Forward primer: 5 '-ATCCCCCGGGAGAGATATCGACACCATGGACAGGG-3 ' (SEQ ID NO.5) reverse primer: 5 '-CCTACCTTAATTAATCAGCTGTCCAGGGCCAGCAGGTCC-3 ' (SEQ IDNO.6)
With XmaI/PacI (available from NEB company) double digestion 1455M gene and recombinant plasmid pSC65gagpol, after enzyme is cut both are connected with T4DNA ligase enzyme (available from NEB company) respectively, be built into transferring plasmid pSC65gagpol-1455M.Identify with restriction enzyme XmaI (available from NEB company) single endonuclease digestion and XmaI/PacI (available from NEB company) double digestion.Enzyme is cut the result and is seen Fig. 5.
2, the structure of vaccinia virus recombinant AIDS vaccine VTT Δ A52L/B8R-GPE
Infect 80-90% CEF (CEF) (MOI=0.01～0.1) in blocks with vaccinia virus recombinant VTT Δ B8R/A52L, 37 ℃ adsorbed after 1 hour, supernatant discarded, and use serum-free Eagle ' s substratum rinsing cell 2 times.According to Invitrogen company transfection reagent box (Lipofectin Reagent2000Cat.18292-011) specification sheets, above-mentioned through the CEF of virus infection cell with transferring plasmid pSC65gagpol-1455M transfection.Cultivate after 48 hours, harvested cell and supernatant are stored in-20 ℃ after the freeze thawing three times.Get 10 μ l transfection liquid and inoculate 80% CEF cell in blocks, after 48 hours with nutrient agar medium (Eagle ' s that contains 10mg/mL X-gal, 1% toluylene red, 1% low melting-point agarose) shop spot, the isolated locus coeruleus virus of picking at random, 5 generations of continuous single spot purifying.Extract recombinant virus dna and adopt PCR method to identify, wherein use following primer:
Forward primer: 5 '-TAACGTGATGGATATATTAAAGTCG-3 ' (SEQ ID NO.7)
Reverse primer: 5 '-AACGACACAACATCCATTTTTAAG-3 ' (SEQ ID NO.8)
The result shows that vaccinia virus recombinant amplifies the TK district specific fragment about 9931bp, confirms that the TK district of vaccinia virus recombinant has inserted LacZ gene and HIV goal gene and successfully lacked the TK gene simultaneously.Vaccinia virus recombinant called after VTT Δ A52L/B8R-GPE.The PCR qualification result is as shown in Figure 6.
Western Blot result is as shown in Figure 7, and gagpol and 1455M gene all have expression, and the Gag molecular weight of albumen is about 55KDa, also has the degraded band of a treaty 41KDa in addition, and the proteic size of gp1455M is 140KDa.Experiment is with an anti-immune serum that is respectively with the Balb/c mouse of the env of HIV and gag protein immunization, and two resist and are the sheep anti-mouse igg of HRP mark, available from middle mountain biotech company.
Employing is with quadrat method construction of recombinant virus VTT Δ B8R-gpe.In brief, infect 80-90% former generation CEF (CEF) (MOI=0.01～0.1) in blocks with VTT Δ B8R (preserving number: CGMCC 4714), transferring plasmid pSC65gagpol-1455M transfection is above-mentioned through the CEF of virus infection cell.Cultivate after 48 hours, harvested cell and supernatant are stored in-20 ℃ after the freeze thawing three times.With the continuous single spot purifying 5 generations acquisition recombinant virus VTT Δ B8R-gpe of aforesaid method.Recombinant virus PCR and Western Blot authentication method are the same, and qualification result shows that the TK district of VTT Δ B8R-gpe has inserted LacZ gene and HIV goal gene and successfully lacked TK gene (result does not show) simultaneously.
The propagation of embodiment 4:A52L genetically deficient replicative vaccinia virus vector in different cells
VTT, VTT Δ A52LLacZ, VTT Δ B8R and VTT Δ B8R/A52L are infected generous flask culture 80-90% monolayer in blocks (CEF, Vero and Hela) respectively with the virus quantity of m.o.i=0.02, behind the absorption 1h, rinsing 2 times.In 37 ℃, at 5%CO
2Cultivate in the incubator of concentration, respectively at harvested cell behind 2h, 12h, 24h, 36h, 48h and the 72h, freeze thawing 3 times is measured virus titer simultaneously and is drawn growth curve (Fig. 8 A-8C) in each cell.The result shows: A52L, B8R gene separately or the associating deletion mutantion propagation of replicative vaccinia virus vector in cell in vitro is not had remarkably influenced.
Embodiment 5:A52L/B8R genetically deficient is to replicability vaccinia virus vector and AIDS vaccine Influence on security
1, rabbit skin virulence experiment
Virus VTT, VTT Δ A52LLacZ and VTT Δ A52L/B8R-GPE are respectively with 1 * 10
7, 1 * 10
6, 1 * 10
5, 1 * 10
4Pfu/100 μ L is injected in (2kg) back intracutaneous of the NZw of shaving the light dorsal body setae (available from Beijing rabbit industry plant that increases income).The continuous 14 days red and swollen situation in observed and recorded injection of skin position, and with slide calliper rule survey its infiltration diameter and downright bad diameter.This research is observed 3-4 days and is begun to occur necrosis, and the 7th day downright bad diameter is to peak, and VTT is 10
4Necrosis promptly appears in pfu, and VTT Δ A52L/B8R-GPE to 10
7Just occur downright bad.VTT Δ A52LlacZ does not occur difference preceding half section of observation period with VTT, than VTT dwindling of very fast time is arranged in later stage spot footpath, and the reorganization poison is in observing end of term acne spot all fully recover (result sees Fig. 9 A).Choose 10
7The downright bad diameter statistics of pfu/100 μ L the 7th day, result show that A52L, B8R gene unite deletion and produce significant skin attenuation effect (p＜0.05) (result sees Fig. 9 B).
2, the mouse collunarium virulence experiment of recombinant virus
3 week BALB/c in age (H-2d) female mices (body weight 19-25 gram is available from Nat'l Pharmaceutical & Biological Products Control Institute) are divided into 5 groups, and 5 every group, from 5 * 10 of nasal inoculation dilution
4The VTT of pfu/20 μ L, VTT Δ A52LLacZ and VTT Δ A52L/B8R-GPE establish one group of mouse of PBS intranasal inoculation as control group.Set time every day observes continuously and claims to survey body weight 15 days, and is ordinate zou with the mouse mean body weight of measuring with the variation per-cent of the initial body weight of mouse, with observing time point be that X-coordinate is mapped.The result finds that the VTT group was inoculation the 8th day, and the initial weight loss of body weight is remarkable, and other recombinant virus inoculation group body weight is along with slight decline, and the 10th day body reopened the beginning and returned steadily and increase.The prompting the strain of reconstructing in the collunarium model of mouse, show the attenuation effect.(result sees Figure 10)
3, virulence experiment in the nude mouse of recombinant virus
Use 7-8 BALB/c-nu nude mice in age in week (body weight 16-18 gram is available from Beijing China Fukang biotech inc); If 1 group of saline water (NS) control group, transform each 3 groups of bovine vaccine strain VTT Δ B8R-gpe, VTT Δ A52L/B8R-GPE without vaccinia virus Tiantan strain of transforming (VTT) and series; Every group of 4 nude mices; NS organizes intraperitoneal injection of saline, and VTT respectively organizes respectively abdominal injection and inoculates corresponding venom 1 * 10
2Pfu, 1 * 10
3Pfu and 1 * 10
4Pfu, VTT Δ B8R-gpe and VTT Δ A52L/B8R-GPE respectively organize respectively abdominal injection and inoculate corresponding venom 1 * 10
6Pfu, 1 * 10
7Pfu and 1 * 10
8Pfu.Observe nude mice existence situation every day, observed for 6 weeks continuously, record nude mice death toll.Abdominal cavity inoculation 1 * 10
3Nude mice death appearred on the 8th day in pfu VTT, inoculation 1 * 10
4Pfu VTT, mortality ratio is 100%, and inoculates 1 * 10
5Pfu VTT Δ B8R/A52L only has 25% dead mouse, and results suggest A52L and B8R gene are united deletion has tangible attenuation effect nude mice.VTT Δ B8R-gpe and VTT Δ A52L/B8R-GPE insert in the TK district of recombinant virus VTT Δ B8R and VTT Δ B8R/A52L respectively that HIV is gene constructed to form and since inactivation the TK gene, the virulence of recombinant virus further reduces.Abdominal cavity inoculation 1 * 10
8Pfu and 1 * 10
7Pfu VTT Δ B8R-gpe mortality ratio is respectively 25% and 75%, inoculation 1 * 10
8～1 * 10
6Pfu VTT Δ A52L/B8R-GPE), each group does not have dead in the observation period.The result shows that the VTT Δ B8R-gpe virulence that the VTT Δ A52L/B8R-GPE of A52L and B8R Gene Double disappearance more singly lacks the B8R gene has further reduction.(result sees table 1)
Table 1
Embodiment 6: AIDS vaccine VTT Δ A52L/B8R-GPE is inductive HIV specific immune response in animal body
1, the initial immunity of dna vaccination-VTT Δ A52L/B8R-GPE booster immunization inductive HIV specific immune response
Adopt the prime-boost strategy in the present embodiment, use 6-8 week BALB/c in age (H-2d) female mice (body weight 18-22 gram is available from Nat'l Pharmaceutical & Biological Products Control Institute) to detect the effectiveness of vaccine of the present invention.The mouse tibialis anterior muscle is injected dna vaccination pDRVI1.0-syngpnef and pDRVI1.0-gp145 (by this laboratory is vector construction with pDRVI1.0, expresses HIV CN54gag, nef, pol and gp1455M respectively) 50 μ g/100 μ l respectively.(structure of pDRVI1.0 and as study on the carrier referring to document LIHai-shan LIU Yong LI Ding-feng ZHANG Ran-ran TANG Hai-li ZHANGYu-wei HUANG Wei LIU Ying PENG Hong XU Jian-qing HONG Kun-xueSHAO Yi-ming.Enhancement of DNA vaccine-induced immune responses by a72-bp element from SV40 enhancer, Chinese Medical Journal 2007; 120 (6): 496-502) interval 3 all experimental mice are through subcutaneous vaccination vaccinia virus recombinant (VTT Δ B8R-gpe or VTT Δ A52L/B8R-GPE, immunizing dose 10
6The PFU/ mouse) or the intramuscular injection dna vaccination.
Through solid-phase enzyme-linked immune spot technology (ELISPOT) detect splenocyte external after the HIV antigenic stimulation counting of secretion of gamma-IFN, IL-2 and IL-4 cell: 2 week of last immunity the back put to death mouse; Get mouse spleen and prepare single cell suspension, adjustment cell concn to 1 * 10
7(individual/ml).Press appended description operation with mouse Elispot detection kit (Mouse IFN-γ and Mouse IL-2 and Mouse IL-4Elispot detection kit are available from BD company).After stimulating 24 hours, through reading the cell count (unit is SFU, spotforming unit) that the plate counting produces IFN-γ, IL-2 and IL-4 like the listed stimulator polypeptide of table 2 (final concentration 4 μ g/ml).The result shows; DNA+VTT Δ A52L/B8R-GPE group and DNA+VTT Δ B8R-gpe group have all excited the specific t cell response of intensive HIV; The response intensity of DNA+ VTT Δ A52L/B8R-GPE group is a little more than DNA+VTT Δ B8R-gpe group, but difference does not have significance.Experimental result is seen Figure 11 A, 11B and 11C.Show and adopt DNA just to exempt from-strategy that bovine vaccine is strengthened, VTT Δ A52L/B8R-GPE can induce strong HIV specificity cellular immunity response in the mouse body.
Table 2
| ? | HIV epitope peptide sequence | Epitopic features |
| SEQ?ID?NO.：9 | GKEVHNVWATHACVPTDPNP | H-2 d |
| SEQ?ID?NO.：10 | SELYKYKVVEIKPLGIAPTA | H-2 d |
| SEQ?ID?NO.：11 | QQSNLLRAIEAQQHLLQLTV | H-2 d |
| SEQ?ID?NO.12 | GTVLVGPTPVNIIGR | H-2 d |
| SEQ?ID?NO.13 | VGPTPVNIIGRNLLT | H-2 d |
| SEQ?ID?NO.14 | VHGVYYDPSKDLIAE | H-2 d |
| SEQ?ID?NO.15 | YYDPSKDLIAEIQKQ | H-2 d |
| SEQ?ID?NO.16 | AAMQILKDTINEEAA | H-2 d |
2, VTT Δ A52L/B8R-GPE immunity two pin inductive HIV specific immune responses
6-8 week BALB/c in age (H-2d) female mice 0,3 week through femoribus internus inoculated with subcutaneous injections vaccinia virus recombinant VTT Δ B8R-gpe or VTT Δ A52L/B8R-GPE, immunizing dose 10
6PFU.Control group is according to immune programme for children identical with experimental group and dose inoculation VTT.Respectively at putting to death mouse after 2 weeks, 6 months after the last immunity, get mouse boosting cell, the cell count of secretion of gamma-IFN after the ELISPOT detection HIV antigenic stimulation, method is the same.The result shows: in 2 weeks after the last immunity, experimental group has all excited the comparatively specific t cell response of intensive HIV, with control group significant difference (p＜0.05) is arranged.The cell count of 2 * VTT Δ A52L/B8R-GPE group secretion of gamma-IFN is a little more than 2 * VTT Δ B8R-gpe group, but there was no significant difference (Figure 12) between group.Back 6 months of last immunity, the cell count of experimental group secretion IFN-γ had obvious decline after than the last immunity in 2 weeks, but 2 * VTT Δ A52L/B8R-GPE group is significantly higher than 2 * VTT Δ B8R-gpe, and group difference has statistical significance (p＜0.05) (Figure 13 A).Show and adopt two pin recombinant vaccinia vaccine immunity strategies, VTT Δ A52L/B8R-GPE inductive cellular immunization is more lasting.
With the antigen coated enzyme plate of Env gp120; Indirect elisa method detects mice serum Env specific IgG antibodies; The result shows; The back IgG antibody that still can detect higher titre in 6 months of last immunity, but there was no significant difference between VTT Δ A52L/B8R-GPE and VTT Δ B8R-gpe group explain that VTT Δ A52L/B8R-GPE can induce strong and persistent humoral immunization.Experimental result is seen Figure 13 B.
Experimental result confirms; The vaccinia virus recombinant VTT Δ A52L/B8R-GPE that the present invention makes up; Can in BALB/c (H-2d) mouse body, induce specific cellular immunization of HIV and humoral immunization; Particularly after the initial immunity of dna vaccination, VTT Δ A52L/B8R-GPE booster immunization can significantly improve immunoreactive intensity; And VTT Δ A52L/B8R-GPE continuous immunity two pin inductive cell immune responses can be kept the long time, show certain immunological memory enhanced.
Above embodiment only is used to explain the present invention, and it has no intention scope of the present invention is made any restriction.Obviously, under the situation that does not break away from spirit of the present invention and essence, those skilled in the art can make multiple change and variation to the present invention, and therefore, these changes and variation are same in the scope that the application requires to protect.
Claims (6)
1. replicative vaccinia virus, the A52L gene and the B8R gene of wherein said vaccinia virus gene group lack simultaneously, and have inserted the antigenic polynucleotide of at least a coding HIV in the TK district.
2. the replicative vaccinia virus of claim 1, wherein said replicative vaccinia virus is a vaccinia virus Tiantan strain.
3. claim 1 or 2 replicative vaccinia virus, the antigenic polynucleotide of wherein said at least a coding HIV derive from HIV-1B '/C reorganization hypotype 97CN54 strain.
4. each replicative vaccinia virus among the claim 1-3, wherein said HIV antigen is selected from one or more among env, gag and the pol.
5. each replicative vaccinia virus is used for preventing or treats the purposes of the vaccine composition of individual HIV virus infection among the claim 1-4 in preparation.
6. AIDS vaccine, it comprises among the claim 1-4 each replicative vaccinia virus.
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| CN1754002A (en) * | 2002-08-12 | 2006-03-29 | 大卫·科恩 | Methods and compositions concerning poxviruses and cancer |
| CN101658670A (en) * | 2008-08-25 | 2010-03-03 | 中国疾病预防控制中心性病艾滋病预防控制中心 | AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector |
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| CN1754002A (en) * | 2002-08-12 | 2006-03-29 | 大卫·科恩 | Methods and compositions concerning poxviruses and cancer |
| CN101658670A (en) * | 2008-08-25 | 2010-03-03 | 中国疾病预防控制中心性病艾滋病预防控制中心 | AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector |
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