CN101107359A - A method of prime-boost vaccination - Google Patents

A method of prime-boost vaccination Download PDF

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CN101107359A
CN101107359A CNA2005800405721A CN200580040572A CN101107359A CN 101107359 A CN101107359 A CN 101107359A CN A2005800405721 A CNA2005800405721 A CN A2005800405721A CN 200580040572 A CN200580040572 A CN 200580040572A CN 101107359 A CN101107359 A CN 101107359A
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gag
vaccine
gene
gly
leu
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本多三男
松尾和浩
浜野隆一
泉泰之
D·普罗姆卡哈提乔
K·巴拉钱德拉
R·萨迪泰特
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Department Of Medical Sciences Ministry Of Health Thailand
Japan Science and Technology Agency
National Institute of Infectious Diseases
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Japan Science and Technology Agency
National Institute of Infectious Diseases
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Abstract

As a novel means for effective prevention from HIV-1 CEF01_AE infection, the present invention provide a method of prime-boost vaccination comprising a priming step by a recombinant BCG vaccine and one or more boosting steps by a recombinant vaccine, wherein both of the recombinant BCG vaccine for priming step and the recombinant vaccine for boosting steps have at least one gene of HIV- 1 CRFO 1_AE strain.

Description

The method of prime-boost vaccination
Technical field
The application's invention relates to the method that is used for the syndromic prime-boost vaccination of AIDS that caused by HIV-1 CRF01_AE strain.
Background technology
The fulminant of 1 type human immunodeficiency virus (HIV-I) is propagated the serious problems that become country in Southeast Asia.In Thailand, dying near 1.3 million people's mouthfeels has HIV-I, and it is said that exist near 200000 AIDS patients the end of the year 2002.Report is verified recently, find that CCR5-tropic CRF01_AE virus (Subbarao etc., 2000) is arranged in medication crowd's great majority morbidity case, and present this recombinant forms is in the ascendance in this country.Therefore press for the preventative vaccine that exploitation should be integrated the anti-HIV-1 CRF01_AE of CRF 01_AE deutero-antigen or epi-position.More existing candidate vaccines that are in III phase clinical stage for example AIDSVAX B/E reorganization gp120 (Berman etc., 1999, Migasena etc., 2000) and with the reorganization canary pox virus (canarypoxvirus) of AIDSVAX B/E combination.Yet other vaccine based on carrier also is not used to develop the AIDS vaccine of anti-CRF01_AE up to now.
The attenuation BCG strain (hereinafter, being called " BCG ") of Mycobacterium bovis (Mycobacterium bovis) is the most universal a kind of live vaccine that is used for the people, and it has the low-down risk that causes severe complication.Well-known this vaccine-induced strong and long lasting t helper cell 1 type is replied, and this replys for activating and keeping cytotoxic T cell (CTL) is important.There have been many reports to prove recombinant BCG (rBCG) the vaccine potential immunogenicity (Honda etc., 1995, Lagranderie etc., 1997, Yasutomi etc., 1995) of target HIV-1 and simian immunodeficiency virus (SIV).But, cause that low HIV or SIV specific CTL reply because be used for people's routine dose, so even do not have to use separately the candidate AIDS vaccine of recombinant BCG carrier.
Some contrivers of the application's invention have invented a kind of recombinant BCG vaccine, this vaccine is used at communicable disease, particularly AIDS is syndromic excites vaccine inoculation, and has applied for that name is called the patent application (WO03/097087A1: be called " in first to file 1 " hereinafter) of " BCG Vaccine and Utilization Thereof ".In first to file 1, the purposes of the recombinant vaccinia virus strain DIs that is used for the strengthening vaccine inoculation is disclosed also at this.
Summary of the invention
The purpose of the application's invention is by using and develop the vaccine inoculation strategy that invention in first to file 1 provides new anti-HIV-1 CRF01_AE.For this purpose, the inventor finds when using recombinant BCG as challenging antigen and be used in combination other based on the vaccine of virus vector during as reinforcement antigen, the cellullar immunologic response that can induce very effectively at the enhancing of HIV-1 CRF01_AE based on this, has been finished the present invention.
The application's invention is the method for prime-boost vaccination, comprise the exciting step of using recombinant BCG vaccine and the reinforcement step of one or more use recombiant vaccines, the recombiant vaccine both who wherein is used for the recombinant BCG vaccine of exciting step and is used to strengthen step has at least a gene of HIV-1CRF01_AE strain.
In described invention, preferred embodiment is the gag gene that two kinds of recombiant vaccines all have the HIV-1CRF01_AE strain at least.In this embodiment, the aminoacid sequence of more preferably described gag genes encoding SEQ ID NO:2, and this gag gene has the nucleotide sequence of SEQ IDNO:1 in addition.
In described invention, another embodiment preferred is that the gene of recombinant BCG vaccine is modified, so that the 3rd of each codon is replaced and do not change amino acid by G or C.
In described invention, the recombiant vaccine that is used to strengthen step is that recombinant vaccinia virus strain DIs also is an embodiment preferred.In this embodiment, the preferred reinforcement step that is to use recombinant vaccinia virus strain DIs comprises at least and carrying out twice.
In described invention, it also is further preferred embodiment that vaccine is used with the dosage of each experimenter 0.05-0.1mg.
Invention in addition is the gag gene of HIV-1 CRF 01_AE strain, and the recombinant BCG vaccine with described gag gene, and wherein said gag gene has the nucleotide sequence of SEQ ID NO:1.
What still invent in addition is the Gag albumen and the proteic antibody of the described Gag of specific recognition of HIV-1 CRF01_AE strain, and wherein said Gag albumen is described gag expression of gene product.
Term of the present invention and notion can specifically definition in the description of embodiment of the present invention and embodiment.And being used to implement multiple technologies of the present invention can be by those skilled in the art according to known publication etc. easily and operation reliably, has quoted except the special technique in source.For example, at Remington ' s Pharmaceutical Sciences, 18th Edition, ed.A.Gennaro, Mack Publishing Co., Easton, PA, the preparation of the vaccine of describing in 1990, and at Sambrook and Maniatis, in Molecular Cloning-ALaboratory Manual, Cold Spring Harbor Laboratory Press, NewYork, 1989; Ausubel, F.M. etc., Current Protocols in MolecularBiology, John Wiley﹠amp; Sons, New York, N.Y., genetically engineered and the Protocols in Molecular Biology described in 1995, or the like.
In addition, the invention of listing hereinbefore is to finish by developing described invention at first to file 1-3, and all at first to file 1-3 by introducing the present invention as a reference.
Therefore, according to the inventive method, the HIV-I antigen-specific immune response can be able to effective enhancing in animal model.
Description of drawings
Fig. 1. be the structure iron of the HIV-1 CRF 01_AE Gag expression vector among the BCG and rBCG clone's protein immunoblot analytical results figure.The arrow of hsp60 promotor is represented transcriptional orientation.Km, Ori-mycobacterium and MCS represent the Kans gene respectively, contain the dna fragmentation and the multiple clone site of the replication orgin of the mycobacterium that comes from pAL 5000 plasmids.With the Gag antigen classification on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the rBCG product of cell lysis, be transferred on the nitrocellulose filter and and develop with the monoclonal antibody of anti-HIV-1 Gag p24.
Fig. 2. be to express the structure iron of the antigenic rDIs virus of HIV-1CRF01_AE Gag and the protein immunoblot analytical results figure of the CEF product of cell lysis that rDIs infects.VV-DIs, p11 and p 7.5 represent non-reorganization DIs genome, Smallpox Vaccine p11 and p 7.5 promoter genes respectively.Analyze SIV Gag antigen in the CEF product of cell lysis that rDIs infects by the same procedure in the legend of Fig. 1, described.
Fig. 3. by each CTL activity of bringing out in 1 month with 0.1mg rBCG-GagE subcutaneous injection immunity BALB/c mouse.Separating Morr. cell, stimulate splenocyte again with every group of 5 series peptides, with its action effect device cell, described series peptide result produces 10 groups of 10 different zones that are positioned at complete HIV-1 gag, also uses the 51Cr mark as target cell with the recombinant vaccinia virus infection that contains HIV-1gag in the P815 cell simultaneously.1-10 represents specificity cracking percentage at every group of peptide with numbering, and the specificity cracking percentage of corresponding cell that will inject the mouse of rBCG/pSO246 simultaneously contrasts as immunity.
Fig. 4. this histogram graph representation through the splenocyte of rBCG/HIV-1 gag immunity at the stimulation of carrying out with the different HIV-1Gag peptide group that is positioned at 10 gag peptide zones again, at effector: the target ratio is 100: 1 o'clock a specificity cracking percentage.
Fig. 5 be illustrated in rBCG-GagE excite and the monkey of strengthening with rDIs-GagE in the ELISpot activity of interferon-gamma.Carried out intracutaneous at the 0th day with rBCG-GagE (0.1mg) and excite, and the 10th week after exciting and the 15th all rDIs-GagE (10 that uses 7Pfu) strengthen twice.After Gag antigenic stimulation, calculate the number of the peripheral blood lymphocytes of secretion interferon-gamma with conventional test kit with the CRF0 1_AE that recombinates.
That Fig. 6 represents is specific to SIV Gag, produce the flow cytometry of the CD8+T cell of IFN-γ.Carry out the vitro culture of the PBMC of macaque with the peptide that overlaps, and with intracellular IFN-γ dyeing.
Embodiment
The present invention is the method for prime-boost vaccination, comprise the exciting step of using recombinant BCG vaccine and the reinforcement step of one or more use recombiant vaccines, and the feature of this method of vaccination is: the recombiant vaccine both who is used for the recombinant BCG vaccine of exciting step and is used to strengthen step has at least a gene of HIV-1 CRF01_AE strain.
In the method, recombinant BCG vaccine comprises the recombinant BCG as activeconstituents.Recombinant BCG is the BCG strain that transforms with at least a expression carrier with HIV-1 CRF01_AE strain.For the BCG strain, can use the BCG strain of the known practical vaccine inoculation that is used for treating pulmonery tuberculosis.For expression vector, can use the carrier (for example plasmid pSO246) of the mycobacterium that has been used to prepare conventional recombinant BCG vaccine.Gene that can be by inserting HIV-1 CRF01_AE to the cloning site of this carrier makes up described expression vector.And, those promotors and the terminator sequence that come from any promotor and the terminator sequence (for example coming from the promotor and the terminator sequence of the heat shock protein(HSP) (hsp) of BCG) of BCG strain and/or come from other mycobacterium strain are connected to described gene, based on this, in BCG, express well from the gene product of HIV-1 CRF01-AE.
The gene that is used for being inserted into the BCG strain is the polynucleotide of coding from the antigen protein of any HIV-1 CRF01_AE strain.More specifically be the antigen protein of HIV-1 CRF01_AE: gag precursor p55, p24 albumen, env albumen gp l20, gp 160 or gp41, pol albumen reversed transcriptive enzyme, nef albumen, tat albumen etc.In these genes, more preferably gag gene product (SEQID NO:2).Particularly, expression vector can make up in pSO246 by the polynucleotide that insert SEQ ID NO:1.
About obtaining the method for antigen gene, the important sequence that will be used for this method cuts out by the plasmid cDNA of suitable restriction enzyme from HIV-1 CRF01_AE pnca gene group or clone.Perhaps, its can use the DNA that comes from HIV-1 CRF01_AE strain infected animals cell or RNA as masterplate, use the primer of proper sequence to come to increase by polymerase chain reaction (PCR).
As the antigen gene of recombinant BCG, the invention provides the gag gene of HIV-1 CRF01_AE strain, it contains the nucleotide sequence of SEQ ID NO:1.The present invention further provides the recombinant BCG vaccine of the gag gene that contains HIV-1 CRF 01_AE strain.
For example calcium chloride method or electroporation are introduced in the BCG strain by currently known methods with the expression vector that as above makes up; and confirm polypeptide expression by western blotting or by known immunizing dose method (for example ELISA), thereby prepare recombinant BCG of the present invention.
When thus the recombinant BCG of preparation when being suspended in the liquid vehicle similar to the carrier of conventional BCG vaccine, the vaccine that recombinant BCG vaccine can prepare and obtain can be actually used in immune induction.
The preferred embodiments of the invention are to modify the insertion gene of recombinant BCG vaccine, so that the 3rd of each codon replaced by G or C and do not change amino acid.Replacement in each codon is shown in (" best codon " this hurdle) in the table 1 with specific form.That is, for example, there is the codon of four kinds of glycine (Gly) that are used to encode: GGT, GGC, GCA and GGG.The Gly codon that meets above-mentioned standard is GGC or GGG.Therefore, the Gly codon in the aminoacid sequence of some antigen protein is GGT or GGA, and the 3rd T (thymus pyrimidine) or A (VITAMIN B4) are replaced with C or G.
Table 1
Amino acid Codon Best codon
Glycine L-Ala Xie Ansuan leucine Isoleucine serine threonine halfcystine methionine(Met) l-asparagine glutamine phenylalanine tyrosine tryptophane aspartic acid L-glutamic acid Histidine Methionin arginine proline(Pro) Gly Ala Val Leu Ile Ser Thr Cys Met Asn Gln Phe Tyr Trp Asp Glu His Lys Arg Pro G A V L I S T C M N Q F Y W D E H K R P GGT、GGC、GGA、GGG GCT、GCC、GCA、GCG GTT、GTC、GTA、GTG CTT、CTC、CTA、CTG、TTA TTG ATT、ATC、ATA TCT、TCC、TCA、TCG、AGT AGC ACT、ACC、ACA、ACG TGT、TGC ATG AAT、AAC CAA、CAG TTT、TTC TAT、TAC TGG GAT、GAC GAA、GAG CAT、CAC AAA、AAG CGT、CGC、CGA、CGG、AGA AGG CCT、CCC、CCA、CCG GGC、GGG GCC、GCG GTC、GTG CTC、CTG、TTG ATC TCC、TCG、AGC ACC、ACG TGC ATG AAC CAG TTC TAC TGG GAC GAG CAC AAG CGC、CGG、AGG CCC、CCG
In the present invention, preference pattern is all positions in each codon of displacement, so that comprise G or C as much as possible under by the unaltered condition of this codon amino acids coding residue type.Such replacement can be applied to leucine (Lue) and arginine (Arg).That is to say, in the best codon that is displayed in Table 1, preferably select CTC or CTG as the Leu codon rather than contain the codon (TTG) of two " T ".In addition, preferably select CGC or CGG as the Arg codon rather than contain the codon (AGG) of " A ".
The codon replacement is based on following discovery as described above.That is, known BCG genome is made up of the DNA with high G+C content and the 3rd of codon is preferably GC strongly to (J.Virol.75:9201-9209,2001; Infect.Immun.57:283-288,1989).In addition, according to the information (Nucl.Acids Res.28:292,2000) of relevant BCG gene of accumulation, know also that the TTA codon of the AGA codon of Arg and Leu seldom uses (be respectively whole codons 0.9% and 1.6%).On the other hand, for example, known HIV-I is that AT is right at the 3rd of codon preferably.In other words, in the encoding sequence of HIV-1p24 gene, 9/11 Arg codon uses AGA, and 6/18 Leu codon uses TTA.As everyone knows, amount relevant (Nature 325:728-730,1987 of corresponding aminoacyl-tRNA in the preference of codon usage frequency and the unicellular organism body; Mol.Biol.Evol.2:13-34,1985).The amount that it is believed that aminoacyl-tRNA corresponding with Arg codon (AGA) and Leu codon (TTA) (it is preferred for the HIV-1p24 gene) in the BCG cell is low-down.
Therefore, in this embodiment preferred, the gene design that will be used for being inserted into recombinant BCG becomes such base sequence, described base sequence conforms to (promptly with the particularly preferred codon usage frequency of BCG cell, the 3rd of codon is G or C, and this codon contains G as much as possible or C in addition).
Be introduced in the gene in order to replace corresponding to the preferred base of each codon, can adopt Kunkel method (the Proc.Natl.Acad.Sci.USA 82:488 that knows, 1985 and Methods in Enzymology 154:367,1987), the method for knowing is for example used the method, sudden change-induction type PCR method of sudden change test kit etc.
For second composition of of the present invention exciting-reinforcement, can use any reinforcement antigen of expressing the antigen gene identical with above-mentioned recombinant BCG vaccine.For example adenovirus, poliovirus, influenza virus, rhinovirus (rhinovirus), varicella virus, vaccinia virus, salmonella (Salmonella) and listeria bacteria (Listeria) plant can to use recombinant viral vector with polypeptide gene identical with recombinant BCG vaccine (exciting vaccine) and bacteria carrier.Especially, recombinant vaccinia virus strain DIs (in first to file 2) is preferred strengthening vaccine.Further preferably using the reinforcement step of recombinant vaccinia virus strain DIs to comprise at least carries out twice.
Excite using of vaccine and strengthening vaccine for example to inject or dosage forms for oral administration is finished by currently known methods.Although dosage, route of administration and dosage regimen may depend on will test individual classification (human or animal), body weight, want inductive immunization type etc., for example exciting vaccine can be 0.05-1mg and strengthening vaccine can be 10 5To 10 10Individual plaque forming unit.The timed interval between twice vaccine inoculation can be 2-12 month.
The present invention further provides the gag gene of HIV-1 CRF01_AE strain, described gag gene contains the nucleotide sequence of SEQ ID NO:1, and the recombinant BCG vaccine that contains described gag gene, described vaccine is preserved in IPOD according to budapest treaty, and preserving number is BP-0000.
Gag gene of the present invention is different from known gag gene shown in Figure 7, and therefore it is new gene.Thereby new gag gene of the present invention has following effectiveness.
Gag gene of the present invention can be used as insertion sequence, and described insertion sequence is used to make up the recombinant BCG that is used to excite vaccine, and can be used for making up for example recombinant vaccinia seedling DIs of strengthening vaccine.One of ordinary skill in the art can be passed through, and for example uses suitable restriction enzyme or PCR method to come to obtain described gag gene from the BCG of preservation.About PCR method, can use one group of primer describing as among the embodiment 1, promptly have those primers of SEQ ID NO:3 and 4.
Gag gene of the present invention can also be used to diagnose AIDS patient whether to infect the HIV-1CRF01_AE strain.This diagnosis can be for example by directly to from the isolating gag gene sequencing of patient and this sequence and SEQ ID NO.1 sequence of the present invention are relatively implemented.Perhaps, can adopt one group of PCR method of use to diagnose to the specific primer of SEQ ID NO:1.That is, if the patient infection HIV-1 CRF01_AE strain, then the gag gene of this uniqueness is increased by PCR method.In addition, this diagnosis can be used the dna microarray system implementation, and described dna microarray system contains the sequence-specific probe to SEQ ID NO:1.
Gag gene of the present invention can be further used for producing the Gag albumen of HIV-1 CRF01_AE strain, and described Gag albumen also is an invention provided by the invention.Gag albumen of the present invention can be used as target protein, and described target protein is used to develop anti-AIDS, particularly the pharmaceutical preparation of the disease of anti-HIV-1 CRF01_AE strain infection.Gag albumen also can be used to develop the diagnostic reagent of the disease that HIV-1 CRF01_AE strain infects.For example, use Gag albumen will produce the antibody of anti-HIV-1 CRF01_AE strain as immunogen.
Gag albumen of the present invention, the polypeptide that promptly has the aminoacid sequence of SEQ ID NO:2 can be by the preparation of following currently known methods: for example based on the chemosynthesis of the peptide of aminoacid sequence provided by the invention, or use the DNA recombinant technology of gag gene provided by the invention.For example, with the DNA recombinant technology, prepare under the proteic situation of Gag by in appropriate host-carrier system, expressing the gag gene, might in intestinal bacteria, subtilis, yeast, insect cell, zooblast etc., obtain a large amount of Gag albumen.
Antibody of the present invention is polyclonal antibody or monoclonal antibody, and it comprises hole molecule (hole molecule), Fab, the F (ab ') that is bonded to Gag albumen epi-position 2, the Fv fragment.Polyclonal antibody can from Gag albumen or its partial peptide immunity the serum of animal obtain.Monoclonal antibody can be according to " Monoclonal Antibody " James W.Goding, thirdedition, and Academic Press, the currently known methods of describing in 1996 obtains.
To come the more detailed the present invention of specifically describing by the following example hereinafter now, but the present invention is not limited by embodiment.
Embodiment
The structure of expression vector
Dna fragmentation (Thole etc., the 1987) clone of the hsp60 gene of coding BCG is advanced in the SmaI-SalI site (pUC-hsp60) of pUC18.To advance in the MunI-KpnI site of pUC-hsp60 corresponding to the multiple clone site of hsp60 gene and the synthetic DNA fragment cloning in termination subarea, and insert the KpnI catenation sequence in the EcoRI site subsequently, produce the pUC-hspK carrier.To increase from Thailand patient's PBMC by PCR from the gag gene of HIV-1 CRF01_AE clinical separation strain M33.The primer that uses is as follows:
Forward primer: 5 '-ATATATCAATTGATCTAGCGGAGGCTAGAAGGAGAGAG-3 ' (SEQ ID NO:3)
Reverse primer: 5 '-ATATAATGGATCCCTAATACTGTATCATCTGC TCCTGTAT-3 ' (SEQ ID NO:4).
MunI-BamHI is cut the same loci that the PCR product cloning is advanced the pUC-hspK carrier, obtain pUC-hsp-gagE.Cut this plasmid and the small segment subclone is advanced generation pSO-gagE (Figure 1A) among the pSO246 (Matsumoto etc., 1994) with KpnI.
The conversion of BCG Tokyo strain
The seed lot of BCG Tokyo substrain is seeded in the 50ml 7 H9-ADC meat soups and 37 ℃ of following joltings hatched 14 days.Culture is mixed with aseptic 50% glycerine, and suspendible is prepared the 1ml BCG solution of 100 equal portions, and is preserved down in-80 ℃ subsequently.One equal portions BCG liquid storage is inoculated in the 7H9-ADC meat soup of 100ml into and and hatched 10 days 37 ℃ of following joltings.Under 3000rpm, collected the BCG cell in centrifugal 5 minutes and it is suspended in the 10% cold glycerine of 10ml.Under 2500rpm after centrifugal 5 minutes, with BCG cell resuspending in cold 10% the glycerine of 5ml.Repeat this step twice.At last, with BCG cell resuspending in the glycerine of 2ml 10%.Take out the BCG cell solution of 100 μ l and it is mixed with the 2 μ g expression plasmid pSO-gagE of the Xiao Chi that is used for Gene-Pulser (Bio-Rad) (gap of 0.2cm).Under 2500V, 25 μ F and 1000ohm, carry out electroporation.Cell cooled on ice 5 minutes, is added the 7H9 meat soup of 150 μ l and hatched under 37 ℃ 2 hours.The BCG cell coated on the 7H10-agar plate that contains 20 μ g/ml kantlex and under 37 ℃ hatched for 3 weeks.
The expression of GagE antigen in BCG
Collect the transformant of BCG, it was cultivated for 2 weeks on the new 7H10-agar plate that contains 20 μ g/ml kantlex, be allowed to condition at 2 weeks of growth in the 30ml7H9-ADC meat soup that contains 20 μ g/ml kantlex then.Collect the culture of 2ml,, and subsequently it is carried out supersound process in the TBS of 200 μ l with Tris buffer saline (TBS) washed twice of 0.5ml.10 μ l cell ultrasonication things are used for the sodium lauryl sulphate gel electrophoresis, and this electrophoresis carries out with Multi-Gel4/20 (Daiichi Chemical company, Japan).Fractionated protein electroblotting on nitrocellulose filter, with corresponding monoclonal antibody reactive, and is made its development with the substrate of peroxidase (3,3 '-diaminobenzidine).Successfully obtain to express the antigenic rBCG clone of GagE at last and be referred to as rBCG-GagE (Figure 1B).The structure of the transfer vector of gagE gene on the other hand, we have obtained the antigenic rDIs virus of generation CRF01_AE Gag in infected chick embryo fibroblast (CEF).In brief, gag E gene is cut out from pUC-hspK-gagE by MunI-BamHI, with its with flat endization of Klenow fragment also subsequently subclone to the SmaI site (Ishii etc., 2002) of pUC-vvp 7.5 H.Cut out gag E genetic expression unit and subclone to pUC-DIs carrier (Ishii etc., 2002) with Hind III.The plasmid that obtains is called pUC-DIs-gagE.
The preparation of CEF
From 8 the biggest eggs (10 eggs), obtain the embryo.After in PBS, removing eyes, brain and internal organ, the embryo is cut into small pieces, its EDTA-PBS with 50ml0.02% in the 50ml test tube is handled by seizer.Under 2000rpm, after centrifugal 5 minutes, will also mix lightly 30 minutes in the 100ml PBS-pancreatin (0.05%) of cell suspension in the 500ml flask with aseptic magnetic stirring apparatus.Take out the 50ml supernatant liquor and be poured among the freezing FBS of 10ml by decant(-ation).Cell suspension is added 50ml PBS-pancreatin solution and stirred 30 minutes.Repeat this step 4 time.Allow the cell suspension that amounts to 300ml by aseptic mesh screen removing cell debris, with its under 2000rpm centrifugal 5 minutes, and resuspending was in the Eagle ' of the 500ml that is supplemented with 5%FBS s improvement basic medium (MEM-5%FBS).With this suspension, its every milliliter has about 1 * 10 6Individual cell is poured in the plastic board (big 10ml, little 3ml) and hatches 3 days up to obtaining the CEF individual layer under 37 ℃.
Vaccinia virus recombinant DIs strain makes up
The CEF individual layer of substratum from the 7cm plate removed and adds rDIs-lacZ virus (Ishii etc., 2002) solution (2 * 10 among the MEM-1%FBS of 0.4ml 6And jiggled totally 1 hour in per 20 minutes pfu).Add fresh MEM-5%FBS substratum (2ml) and overnight incubation.Use Clonfectin (Clontech Co.Ltd) that the pUC-DIs-gagE plasmid transfection is advanced among the CEF of rDIs-lacZ infection, locate homologous recombination to gag E gene at the main deletion segment (Fig. 2 A) of parent vaccinia virus DIs strain to cause the lacZ gene.After hatching 3 days, collecting cell and supernatant liquor repeat freezing and thaw twice and be equal cell plastid with supersound process.The viral solution of serial dilution is used for infecting freshly prepd CEF and it was hatched 3 days at the MEM-5%FBS substratum.After removing substratum, on the MEM substratum-agar plate that contains 0.004%5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal), select colourless bacterial plaque.The composition of the substratum that uses is: 1.2% agar among the MEM, 0.225%NaHCO 3, the X-gal of 0.0292%L-Gln, 80 μ g/ml, the kantlex of 40 μ g/ml.Collect the agar fritter of colourless bacterial plaque and be placed in the MEM-1%FBS substratum of the 1ml in the eppendorf pipe by pasteur pipet.Freezing and thaw after, with agar fritter supersound process 5 minutes and under-80 ℃, preserve or be used to infect new CEF.Repeating this indigo plant-hickie selects step 3 time or 4 bacterial plaques all in a flat board all to be shown as colourless.Collecting cell and supernatant liquor from the flat board of a 7cm diameter made its homogenate in 10 minutes by supersound process, and were used for infecting the fresh CEF of 10cm flat board.In perfect medium after hatching 3 days under 37 ℃, collecting cell, with the PBS washed twice and in 0.5ml PBS supersound process 10 minutes with the preparation product of cell lysis.
The expression of Gag E antigen in the CEF that rDIs infects
The SDS-PAGE that is used to use (DaiichiChemical company, Japan) with Multi-Gel 4/20 to carry out the product of cell lysis of 10 μ l.With fractionated protein electroblotting on nitrocellulose filter, with itself and anti-HIV gag V107 monoclonal antibody reactive (Matsuo etc., 1994).With anti-mouse IgG reaction as the peroxidase conjugated of second antibody after, with the substrate of peroxidase (3,3 '-diaminobenzidine) reactive protein is developed.According to the Western engram analysis, obtained in CEF, to express the rDIs clone of gag E gene and be referred to as rDIs-GagE (Fig. 2 B).
The purifying of reorganization DIs virus
The CEF product of cell lysis that rDIs-GagE is infected is used at 20 75cm 2Flask in infect new CEF individual layer and it hatched under 37 ℃ 3 days.After the collection, cell and supernatant liquor is freezing and thaw twice, and excusing from death is handled 10 minutes twice with the homogenate cell, and is used for purifying subsequently.Purification step is as follows: (i) supernatant liquor that 38ml is cultivated placed each test tube (on 36% the sucrose solution of the 5ml of BECKMAN Ultra-Clear1 * 3.5in.25 * 89mm) and under 14000rpm centrifugal 80 minutes gently.(ii) with 5ml, 25ml and use the PBS resuspending throw out (3 times) of 8ml then, place gently on 36% the sucrose solution of 5ml of centrifuge tube, and under 17000rpm centrifugal 30 minutes subsequently.(iii), it is suspended among the PBS of 100ml fully, and separately places crio test tube (every test tube 1ml), and subsequently they are preserved (first) down at-80 ℃ with PBS washing precipitate gently.
Reorganization DIs titration of virus
With CEF in 37 ℃ of 48 porocyte culture plates (2.5 * 10 in CO2gas incubator 5Individual cells/well) cultivated 3 days in.The viral dilution liquid of 10 times of serial dilutions among the MEM-1%FBS is infected CEF (45 μ l/ hole).After hatching 1 hour, the adding fresh MEM-5%FBS substratum of 0.5ml was also hatched 3 days.After removing substratum, 5% formalin solution (among PBSs) of cell in 200 μ l/ holes fixedly spent the night under room temperature.0.02% the methylene blue solution (in PBS) of at room temperature using 200 μ l/ holes is described cell dyeing 3 hours, with the PBS washing and calculate the CPE number subsequently so that calculate virus titer.First batch titre is about 10 5Pfu/ml.For obtaining the rDIs-GagE of higher titre, as described above, with 20 75cm of first batch virus infection 2Fresh CEF cell in the flask is with its cultivation, purifying.Titre with rDIs-GagE virus is adjusted to 1 * 10 at last 8Pfu/ml.
Cellullar immunologic response in the mouse
Being seeded in of rBCG-GagE to BALB/c brought out peptide-antigen-specific CTL (Fig. 3 and 4) in the immunized animal.The rDIs-GagE construct has also brought out very high CTL and has replied in mouse, and proves that clearly it is a high immunogenicity, although its reproducible not.Therefore, our decision in macaque assessment these be used to excite-candidate vaccine of enhancement method.
The enhanced cell immunne response that in monkey, causes by prime-boost vaccination
Excite and use rDIs-GagE (10 by interferon-gamma-ELISpot detection method assessment with rBCG-GagE (0.1mg is through intradermal injection) 7Pfu, intradermal injection ground) the enhanced cell immunne response that causes in macaque of the scheme of strengthening, wherein said rBCG-GagE and rDIs-GagE both are people's actual dose and route of administration.As shown in Figure 5, the double injection of rDIs-strengthening vaccine has strengthened the specific ELISpot of HIV-1Gag very effectively and has replied.Carried out the IFN γ ELISPOT detection method of virus-specific.In brief, 96 hole flat undersides (U-CyTech-BV, Utrecht, Holland) are spent the night with anti-IFN γ mAb MD-1 (U-CyTech-BV) bag under 4 ℃.Wash this plate with the PBS that contains 0.05%Tween 20 (PBST) then and use the PBS that contains 2% bovine serum albumin (PBSA) to seal 1 hour down at 37 ℃.Discard PBSA from this plate.((AIDSResearch and Reference Reagent Program) adds among the PBMC of fresh separated with con A (ConA) or 0.2 μ M blended Gag peptide, and in the flat board of anti-IFN γ bag quilt, in 5%CO2, under 37 ℃, hatched 16 hours then, use ice-cold deionized water cracking subsequently.After washing this plate, (every hole 1 μ g U-CyTech-BV), and was further hatched under 37 ℃ 1 hour to add the biotinylated detection antibody of rabbit anti-IFN γ polyclone.Wash this plate with PBST then, add antibiotin immunoglobulin G (GABA) solution (U-CyTech-BV) of 50 μ l gold mark subsequently and under 37 ℃, hatched 1 hour.After the PBST washing, add activating mixtures (every hole 30 μ l; U-CyTech-BV) and allow this plate colour developing 15 minutes.
(Carl Zeiss Germany) makes cell imaging and calculate the number that spot forms cell (SFC) with KS ELISPOT compact system.SCF is defined as the big stain with fuzzy edge.In order to measure conspicuous level, use for the mean value and the standard deviation of the SFC number of each peptide and set up the baseline that is used for each peptide.Measure corresponding this mean value then and add that the threshold of two standard deviations significantly is worth.If the threshold conspicuous level that has outnumbered the sample that does not have peptide of SCF would think the reaction be male.
And cell within a cell factor dyeing detection method has proved CD8 in by the monkey of immunity +The CTL activated strengthens (Fig. 6).These data declarations, effectively the positive cell immunity can be induced and strengthened by this exciting-reinforcement scheme.In order to detect intracellular IFN by flow cytometer, with the PBMC (5 * 10 of fresh separated 5To 1 * 10 6Individual cell) be suspended in the R-10 substratum and at 37 ℃ down with 5% CO 2Hatched 16 hours with antigen.At last 6-8 hour, add 10 μ g/ml Brefeldin A (Sigma Chemical Co., St.Louis, MO).(SanDiego is CA) as the secondary stimulus molecule for 1 μ g/ml, BD Pharmingen also to add the antibody of CD28 in the process of hatching.After the stimulation, the anti-CD3 (FN18 that washs described cell and put together with fluorescein isothiocyanate (FITC); Biosource, Camarillo, CA) and the anti-CD8 antibody (Leu-2a that puts together of peridinin chlorophyl albumen (Per CP); Becton Dickinson) dyeing.(Becton Dickinson Biosciences, San Jose CA) were hatched 10 minutes and were hatched in addition 10 minutes with FACS percolating solution (Becton Dickinson) to use fluorescence-activated cell sorter (FACS) cracked solution then successively.Wash described cell, the anti-people IFN-gamma antibodies (4S.B3 that puts together with phycoerythrin (PE); BDPharmingen) dye, and fix with 2% Paraformaldehyde 96.Use Cell Quest software (Becton Dickinson) FACS Callibur (Becton Dickinson) analytic sample.
Industrial applicibility
Set forth such as top institute comprehensively, use the recombinant BCG with the strengthening vaccine of determining such as vaccinia virus recombinant combination, can obtain effective immune induction for anti-HIV-1 CRF01_AE according to the application's invention. The present invention will obtain the effective prevention to the HIV-1CEF01_AE infection.
Sequence table
<110>Ministry of public Heslth in Thailand and Japan Science and Technology Agency
＜120〉method of prime-boost vaccination
<130>05-F-078PCT
<150>JP2004-341283
<151>2004-11-25
<160>2
<170>PatentIn version 3.1
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＜213〉1 type human immunodeficiency virus (Human immunodeficiency virus type 1)
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atg ggt gcg aga gcg tca gta tta agt ggg gga aaa tta gat gca tgg 48
Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp
1 5 10 15
gaa aaa att cgg tta cgg cca ggg gga aag aaa aaa tat agg atg aaa 96
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys
20 25 30
cat tta gta tgg gca agc aga gag tta gaa aga ttc gca ctc aac cct 144
His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro
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ggc ctt tta gaa aca gca gaa gga tgt caa caa ata ata gaa cag tta 192
Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile Glu Gln Leu
50 55 60
cag tca aat ctc aag aca gga tca gaa gaa ctt aaa tca tta ttt aat 240
Gln Ser Asn Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn
65 70 75 80
tta ata gta acc ctc tgg tgc gta cac caa agg ata gag gta aaa gac 288
Leu Ile Val Thr Leu Trp Cys Val His Gln Arg Ile Glu Val Lys Asp
85 90 95
acc aag gaa gct tta gat aaa ata gag gaa gta caa aat aag agc cag 336
Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln
100 105 110
caa aag aca cag cag gca aca act gac aca gga agc agc agc aaa gtc 384
Gln Lys Thr Gln Gln Ala Thr Thr Asp Thr Gly Ser Ser Ser Lys Val
115 120 125
agc caa aat tac cct ata gtg caa aat gca caa ggg caa atg gta cat 432
Ser Gln Asn Tyr Pro Ile Val Gln Asn Ala Gln Gly Gln Met Val His
130 135 140
cag cct tta tca cct aga act ttg aat gcc tgg gta aaa gta gta gaa 480
Gln Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu
145 150 155 160
gaa aag ggt ttt aac cca gaa gta ata ccc atg ttc tca gca tta tca 528
Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser
165 170 175
gag gga gcc acc cca caa gat tta aat atg atg cta aat ata gtg ggg 576
Glu Gly Ala Thr Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly
180 185 190
gga cac cag gca gca atg caa atg tta aaa gaa acc atc aat gag gaa 624
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu
195 200 205
gct gca gaa tgg gat agg gta cac cca gta cat gca ggg cct att cca 672
Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Pro
210 215 220
cca ggc cag atg agg gaa cca agg gga agt gac ata gca gga act act 720
Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr
225 230 235 240
agt acc ctt caa gaa caa ata gga tgg atg aca aac aat cca cct ata 768
Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile
245 250 255
cca gtg gga gac atc tat aaa agg tgg ata atc ctg gga tta aat aaa 816
Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270
ata gta aga atg tat agc cct gtt agc att ttg gac ata aga caa ggg 864
Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly
275 280 285
cca aaa gaa ccc ttc aga gac tat gta gat agg ttc tat aaa act ctt 912
Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu
290 295 300
aga gcg gaa caa gct aca cag gag gta aaa aac tgg atg aca gaa acc 960
Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr
305 310 315 320
ttg cta gtc caa aat gcg aat cca gac tgt aag tcc att cta aaa gca 1008
Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys SerIle Leu Lys Ala
325 330 335
tta gga aca gga gct aca tta gaa gaa atg atg aca gca tgc cag gga 1056
Leu Gly Thr Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly
340 345 350
gtg gga gga cct agc cat aaa gca agg gtt ttg gct gag gca atg agc 1104
Val Gly Gly Pro Ser His Lys Ala Arg Val Leu Ala Glu Ala Met Ser
355 360 365
cag gta caa cat gca aat ata atg atg cag aga ggc aat ttt aag ggc 1152
Gln Val Gln His Ala Asn Ile Met Met Gln Arg Gly Asn Phe Lys Gly
370 375 380
cag aaa aga att aag tgc ttc aac tgt ggc aaa gaa gga cac cta gcc 1200
Gln Lys Arg Ile Lys Cys Phe Asn Cys Gly Lys Glu Gly His Leu Ala
385 390 395 400
aga aat tgc agg gcc cct aga aaa aag ggt tgt tgg aaa tgc ggg aag 1248
Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys
405 410 415
gaa gga cat caa atg aaa gac tgc act gag aga cag gct aat ttt tta 1296
Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu
420 425 430
ggg aaa att tgg cct tcc aac aag gga agg ccg ggg aat ttt cct cag 1344
Gly Lys Ile Trp Pro Ser Asn Lys Gly Arg Pro Gly Asn Phe Pro Gln
435 440 445
agc aga cca gag cca aca gcc cca cca gca gaa aac tgg ggg atg ggg 1392
Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Asn Trp Gly Met Gly
450 455 460
gaa gag ata acc tcc tta ctg aag cag gag cag aaa gac aag gaa cat 1440
Glu Glu Ile Thr Ser Leu Leu Lys Gln Glu Gln Lys Asp Lys Glu His
465 470 475 480
cct cct cct tta gtt tcc ctc aaa tca ctc ttt ggc gac gac ccc ttg 1488
Pro Pro Pro Leu Val Ser Leu Lys Ser Leu Phe Gly Asn Asp Pro Leu
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tca cag taa 1497
Ser Gln
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＜213〉1 type human immunodeficiency virus
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Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Lys Leu Asp Ala Trp
1 5 10 15
Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Arg Met Lys
20 25 30
His Leu Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Leu Asn Pro
35 40 45
Gly Leu Leu Glu Thr Ala Glu Gly Cys Gln Gln Ile Ile Glu Gln Leu
50 55 60
Gln Ser Asn Leu Lys Thr Gly Ser Glu Glu Leu Lys Ser Leu Phe Asn
65 70 75 80
Leu Ile Val Thr Leu Trp Cys Val His Gln Arg Ile Glu Val Lys Asp
85 90 95
Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Val Gln Asn Lys Ser Gln
100 105 110
Gln Lys Thr Gln Gln Ala Thr Thr Asp Thr Gly Ser Ser Ser Lys Val
115 120 125
Ser Gln Asn Tyr Pro Ile Val Gln Asn Ala Gln Gly Gln Met Val His
130 135 140
Gln Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu
145 150 155 160
Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser
165 170 175
Glu Gly Ala Thr Pro Gln Asp Leu Asn Met Met Leu Asn Ile Val Gly
180 185 190
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu
195 200 205
Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Pro
210 215 220
Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr
225 230 235 240
Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile
245 250 255
Pro Val Gly Asp Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
260 265 270
Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp Ile Arg Gln Gly
275 280 285
Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu
290 295 300
Arg Ala Glu Gln Ala Thr Gln Glu Val Lys Asn Trp Met Thr Glu Thr
305 310 315 320
Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Ser Ile Leu Lys Ala
325 330 335
Leu Gly Thr Gly Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly
340 345 350
Val Gly Gly Pro Ser His Lys Ala Arg Val Leu Ala Glu Ala Met Ser
355 360 365
Gln Val Gln His Ala Asn Ile Met Met Gln Arg Gly Asn Phe Lys Gly
370 375 380
Gln Lys Arg Ile Lys Cys Phe Asn Cys Gly Lys Glu Gly His Leu Ala
385 390 395 400
Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys Gly Lys
405 410 415
Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn Phe Leu
420 425 430
Gly Lys Ile Trp Pro Ser Asn Lys Gly Arg Pro Gly Asn Phe Pro Gln
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450 455 460
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485 490 495
Ser Gln
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atatatcaat tgatctagcg gaggctagaa ggagagag 38
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atataatgga tccctaatac tgtatcatct gctcctgtat 40

Claims (12)

1. the method for prime-boost vaccination, comprise the exciting step of using recombinant BCG vaccine and the reinforcement step of one or more use recombiant vaccines, the recombiant vaccine both who wherein is used for the recombinant BCG vaccine of exciting step and is used to strengthen step has at least a gene of HIV-1 CRF01_AE strain.
2. according to the process of claim 1 wherein that described two kinds of recombiant vaccines all have the gag gene of HIV-1 CRF01_AE strain at least.
3. according to the method for claim 2, wherein said gag genes encoding Gag protein, described protein has the aminoacid sequence of SEQ ID NO:2.
4. according to the method for claim 3, wherein said gag gene has the nucleotide sequence of SEQ ID NO:1.
5. according to each method among the claim 1-4, the gene of wherein said recombinant BCG vaccine is modified, and the 3rd of each codon the replaces with G or C and do not change amino acid like this.
6. according to the process of claim 1 wherein that the described recombiant vaccine that is used to strengthen step is recombinant vaccinia virus strain DIs.
7. according to the method for claim 6, wherein use the reinforcement step of recombinant vaccinia virus strain DIs to comprise at least and carry out twice.
8. according to each method among the claim 1-7, wherein said vaccine is used with the dosage of each experimenter 0.05-0.1mg.
9.HIV-1 the gag gene of CRF01_AE strain, described gene has the nucleotide sequence of SEQ ID NO:1.
10. recombinant BCG vaccine, described vaccine has the gag gene of claim 9.
11.HIV-1 the Gag protein of CRF01_AE strain, described protein are the gag expression of gene products of claim 9.
12. the proteinic antibody of the Gag of specific recognition claim 11.
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