WO2006057454A1 - A method of prime-boost vaccination - Google Patents

A method of prime-boost vaccination Download PDF

Info

Publication number
WO2006057454A1
WO2006057454A1 PCT/JP2005/022221 JP2005022221W WO2006057454A1 WO 2006057454 A1 WO2006057454 A1 WO 2006057454A1 JP 2005022221 W JP2005022221 W JP 2005022221W WO 2006057454 A1 WO2006057454 A1 WO 2006057454A1
Authority
WO
WIPO (PCT)
Prior art keywords
recombinant
gene
gag
hiv
vaccine
Prior art date
Application number
PCT/JP2005/022221
Other languages
French (fr)
Inventor
Mitsuo Honda
Kazuhiro Matsuo
Takaichi Hamano
Yasuyuki Izumi
Duanthanorm Promkhatkaew
Kruavon Balachandra
Ruengpung Sutthent
Original Assignee
Japan Science And Technology Agency
Japan As Represented By Director General Of National Institute Of Infectious Diseases
Departement Of Medical Sciences, Ministry Of Public Health, Thailamd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Science And Technology Agency, Japan As Represented By Director General Of National Institute Of Infectious Diseases, Departement Of Medical Sciences, Ministry Of Public Health, Thailamd filed Critical Japan Science And Technology Agency
Publication of WO2006057454A1 publication Critical patent/WO2006057454A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention of this application relates to a method of prime-boost vaccination for AIDS syndromes caused by HIV-I CRFO 1_AE strain.
  • HIV-I HIV-I
  • CCR5-tropic CRF01_AE viruses are found in most incidence cases in the drug user cohort (Subbarao et al., 2000) and this recombinant form is now dominant in this country. So there is an urgent need to develop a prophylactic vaccine against HIV-I CRFO 1_AE that should incorporate CRF01_AE-derived antigens or epitopes.
  • AIDSVAX B/E recombinant gpl20 (Berman et ah, 1999, Migasena et at, 2000) and recombinant canarypox virus in combination with AIDSVAX B/E.
  • other vector-based vaccine has not been applied to develop AIDS vaccine against CRFO 1_AE until now.
  • BCG Attenuated BCG strain of Mycobacterium bovis
  • BCG Mycobacterium bovis
  • This vaccine is well known to induce a strong and long-lasting T helper type 1 response, which is important to activate and maintain cytotoxic T lymphocyte (CTL).
  • CTL cytotoxic T lymphocyte
  • rBCG recombinant BCG
  • SIV simian immunodeficiency virus
  • the invention of this application has an object of providing a novel vaccination strategy against HIV-I CRFO 1_AE by utilizing and developing the inventions of the Prior Application 1.
  • the present inventors have found that in the case of using recombinant BCG as a priming antigen in combination with other viral vector-based vaccine as a boosting antigen, quite efficiently enhanced cellular immune response could be induced against HIV-I CRFO 1_AE whereupon the present invention has been achieved.
  • An invention of this application is 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-I CRFO 1_AE strain.
  • both of the recombinant vaccines have at least gag gene of HIV-I CRF01_AE strain.
  • the gag gene encodes the amino acid sequence of SEQ ID NO: 2, and further the gag gene has the nucleotide sequence of SEQ ID NO: 1.
  • the gene of the recombinant BCG vaccine is modified so that a third position of each codon is substituted with G or C without a change of an amino acid.
  • the recombinant vaccine for boosting step is a recombinant vaccinia virus strain DIs.
  • the boosting step with the recombinant vaccinia virus strain DIs comprises at least two trials.
  • the vaccines are administered at the dose of 0.05 to 0.1 mg per subject.
  • gag gene of HIV-I CRF01_AE strain having the nucleotide sequence of SEQ ID NO: 1, and a recombinant BCG vaccine having said gag gene.
  • Gag protein of HIV-I CRFO 1_AE strain which is a expression product of said gag gene, and an antibody specifically recognizing said Gag protein.
  • the HIV-I antigen specific immune responses can be efficiently enhanced in animal models.
  • Fig. 1 Structure of HIV-I CRFO 1_AE Gag expression vector in BCG and western immunoblot analysis of rBCG clone. Arrow of hsp ⁇ O promoter indicates direction of transcription. Km, Ori-mycobacteria, and MCS indicate kanamycin resistance gene, DNA fragment containing replication origin in mycobacteria derived from pAL5000 plasmid, and multi-cloning sites, respectively.
  • the Gag antigen in rBCG cell lysate was fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoreisis, transferred onto nitrocellulose membrane filter and visualized with. anti-HIV-1 Gag p24 monoclonal antibody.
  • Fig. 2 Structure of rDIs virus expressing HIV-I CRF01_AE Gag antigen and western immunoblot analysis of rDIs-infected CEF cell lysate.
  • W-DIs, pll, and p7.5 indicate non-recombinant DIs genome, vaccinia pl l and p7.5 promoter genes, respectively.
  • SIV Gag antigen in rDIs-infected CEF cell lysate was analysed by the same procedure described in Fig. 1 legend.
  • Fig. 3 CTL activities elicited by immunization of BALB/c mice with O. lmg rBCG-GagE s.c. once for 1 month.
  • Spleen cells were isolated and restimulated with each group of 5 serial peptides resulting in ten groups occupying ten different regions of entire HIV-I gag as the effector cells, while P815 cells were infected with recombinant vaccinia virus containing HIV-I gag and 51 Cr-labelled as the target cells.
  • Specific lysis against each peptide group was shown as number 1 to 10, while those of mice injected with rBCG/pSO246 was as immunization control.
  • Fig. 4. The bars indicate % specific lysis at Effector : Target 100: 1 of rBCG/ HIV-I gag immunized spleen cells against restimulation with different HIV-I Gag peptide pools occupying ten gag peptide regions.
  • Fig. 5 Interferon-gamma ELISpot activity in rBCG-GagE-primed and rDIs-GagE-boosted monkeys.
  • rBCG-GagE (0.1 mg) was primed at day 0 intradermally and rDIs-GagE (10 7 pfu) was boosted twice at 10 and 15 weeks post-priming.
  • Fig. 6 Flow cytometric analysis of IFN-gamma-producing CD8 + T cells specific for SIV Gag.
  • PBMC from macaques were cultured in vitro with overlapping peptides and stained for intracellular IFN-gamma.
  • the present invention is 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, and the character of this vaccination method is that both of the recombinant BCG vaccine for priming step and the recombinant vaccine for boosting steps have at least one gene of HIV-I CRFO 1_AE strain.
  • the recombinant BCG vaccine contains a recombinant BCG as an active ingredient.
  • the recombinant BCG is a BCG strain transformed by an expression vector having at least one gene of HIV-I CRFO 1_AE strain.
  • the BCG strain it is possible to use widely known ones which have been used for practical vaccination against tuberculosis.
  • the expression vector it is possible to use a vector for mycobacteria (such as a plasmid pSO246) which has been used for the preparation of conventional recombinant BCG vaccine.
  • the expression vector can be constructed by inserting gene(s) of HIV-I CRFO 1_AE into a cloning site of this vector.
  • any promoter and terminator sequences derived from BCG strain (such as promoter and terminator sequences of heat shock protein (hsp) derived from BCG) and/or those derived from other mycobacterial strains are ligated to the gene(s), whereupon the gene-product(s) from HIV-I CRFOl-AE is well expressed in BCG.
  • hsp heat shock protein
  • a gene for insertion into BCG strain is a polynucleotide which encodes antigenic protein from any HIV-I CRF01_AE strain.
  • gag precursor p55, p24 protein, env protein gpl20, gpl60 or gp41, pol protein reverse transcriptase, nef protein, tat protein, etc. which are antigenic proteins of HIV-I CRFO 1_AE.
  • gag gene product SEQ ID NO: 2
  • an expression vector may be constructed by inserting the polynucleotide of SEQ ID NO: 1 into pSO246.
  • the significant sequence for that is cut out by appropriate restriction enzymes from genome gene of HIV-I CRFO 1_AE strain, or cloned plasmid cDNA.
  • it may be amplified by a polymerase chain reaction (PCR) using primers of appropriate sequences using DNA or RNA derived from animal cells infected with HIV-I CRFO 1_AE strain as a template.
  • PCR polymerase chain reaction
  • the present invention provide gag gene of HIV-I CRFO 1_AE strain, which has the nucleotide sequence of SEQ ID NO: 1.
  • the present invention further provide a recombinant BCG vaccine holding the gag gene HIV-I CRFO 1_AE strain.
  • the expression vector constructed as above is introduced into BCG strain by known methods such as a calcium chloride method or an electr op oration method and expression of the polypeptide is confirmed by western blotting or by known immunological measuring method (such as ELISA) whereby the recombinant BCG of this invention can be prepared.
  • a recombinant BCG vaccine can be prepared and the resulting vaccine is able to be actually used for an immune induction.
  • a preferred embodiment of this invention is that the inserted gene of the recombinant BCG vaccine is modified so that a third position of each codon is substituted with G or C without a change of an amino acid.
  • the substitutions in the respective codons are shown in Table 1 in a concretive manner (the column of "optimal codon"). That is, for example, there are four codons for encoding glycine (GIy): GGT, GGC, GCA, and GGG.
  • the GIy codon agreed with the above criteria is GGC or GGG. Therefore, the GIy codon in the amino acid sequence of some antigenic protein is GGT or GGA, the third T (thymine) or A (adenine) is substituted with C or G.
  • a preferable mode is that all positions in each codon is substituted so as to include G or C as much as possible under the conditions in which the type of an amino acid residue encoded by such a codon is not changed.
  • Such a kind of the substitution can be applied on leucine (Lue) and arginine (Arg). That is, among the optimal codons shown in Table 1 , CTC or CTG is preferably selected as a Leu codon rather than the codon (TTG) containing two "T"s. In addition, CGC or CGG is preferably selected as an Arg codon rather than the codon (AGG) containing "A".
  • the codon substitution as described above is based on the following findings. That is, it is known that the BCG genome consists of DNA with a high G + C contents and the third position of the codon strongly prefers GC pair (J. Virol. 75: 9201-9209, 2001; Infect. Immun. 57: 283-288, 1989). Furthermore, from the accumulated information on BCG genes (Nucl. Acids Res. 28: 292, 2000), it is also known that the AGA codon for Arg and the TTA codon for Leu are less frequently used (0.9% and 1.6% of total codons, respectively). On the other hand, for instance, it is known that HIV-I prefers an AT pair at the third position of the codon.
  • the gene for insertion into recombinant BCG is designed to become a base sequence agreed with the frequency of codon usage particularly preferable for the BCG cell (i.e., the third position of the codon is G or C, and furthermore the codon contains G or C as much as possible).
  • any booster antigen which expresses the same antigenic gene as the above recombinant BCG vaccine can be used.
  • Recombinant viral and bacterial vectors such as adenovirus, poliovirus, influenza virus, rhinovirus, varicella virus, vaccinia virus, Salmonella and Listeria species with the same polypeptide gene as the recombinant BCG vaccine (primer vaccine).
  • a recombinant vaccinia virus strain DIs (Prior Application 2) is a preferred booster vaccine. It is futher preferred that the boosting step with the recombinant vaccinia virus strain DIs comprises at least two trials.
  • Prime and booster vaccines can be carried out by known methods such as injection or oral administration.
  • dose, route and schedule may depend on type (human or animal), body weight, type of the immunity to be induced, etc. of the individual to be examined
  • primer vaccine 0.05 to 1 mg and booster vaccine may be 10 5 to 10 10 plaque forming units for example.
  • the time interval between two inoculations of vaccine may be 2 to 12 months.
  • the present invention further provides gag gene of HIV-I CRFO 1_AE strain that has the nucleotide sequence of SEQ ID NO: 1, and a recombinant BCG vaccine holding the gag gene, which has been deposited under Budapest treaty at IPOD as a deposit number BP-OOOO.
  • gag gene of this invention is different from the known gag gene as shown in Fig. 7, and therefore it is novel gene. Consequently, the novel gag gene of this invention has utilities as follows.
  • the gag gene of this invention can be used as an insert for constructing the recombinant BCG for priming vaccine, and also for constructing the boost vaccine such as the recombinant vaccinia DIs.
  • One of ordinary skill in the art may obtain the gag gene from the deposited BCG by, for example, using appropriate restriction enzymes or PCR method.
  • the PCR method the set of primers as described in Example 1, i.e. those of having SEQ ID NO.:3 and 4, may be used.
  • the gag gene of this invention also can be utilized for diagnosing an
  • the diagnosis may be performed, for example, by directly sequencing the gag gene isolated from the patient and comparing the sequence with that of SEQ ID NO: 1 of this invention.
  • PCR method of using a set of specific primers to the SEQ ID NO: 1 may be employed for the diagnosis. That is, if the patient is infected with the HIV-I
  • the unique gag gene is amplified with the PCR method. Further, the diagnosis can be performed with DNA microarray system that comprises probes specific to the sequence of SEQ ID NO: 1.
  • the gag gene of this invention can be further used for producing Gag protein of HIV-I CRFO 1_AE strain, which is also an invention provided by the present invention.
  • the Gag protein of this invention may be useful as a target protein for developing pharmaceuticals against AIDS, especially the HIV-I CRFO 1_AE strain-infectious disease.
  • the Gag protein may be also used for developing a diagnostic agent for the HIV-I CRFO 1_AE strain-infectious disease. For example, using the Gag protein as an immunogen would make an antibody against the HIV-I CRF01_AE strain.
  • the Gag protein of this invention Le., polypeptide having the amino acid sequence of SEQ ID NO: 2
  • the Gag protein is prepared by means of a recombinant DNA technique, by expressing the gag gene in a suitable host-vector system, it is possible to obtain the Gag protein in a large quantity in Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, etc.
  • the antibody of this invention is polyclonal antibody or monoclonal antibody, and it includes hole molecule binding to epitope of the Gag protein, Fab * F(ab')2% Fv fragment.
  • the polyclonal antibody may be obtained from serum of an animal immunized with the Gag protein or its partial peptide.
  • the monoclonal antibody can be obtained according to the know method as described in "Monoclonal Antibody” James W. Goding, third edition, Academic Press, 1996.
  • a DNA fragment encoding hsp ⁇ O gene of BCG was cloned into Smal-SaU. sites of pUC18 (pUC-hsp60).
  • a synthetic DNA fragment which corresponds to multi-cloning site and terminator region of hsp60 gene was cloned into Munl-Kpnl sites of pUC-hsp60 and then inserted Kpnl linker at EcoRl site giving rise to pUC-hspK vector.
  • the gag gene from HIV-I CRFO 1_AE clinical isolate M33 was amplified by PCR from PBMC of a Thai patient. Used primers were as follows: forward primer:
  • BCG Tokyo substrain was inoculated into 50 ml of 7H9-ADC broth and cultured at 37°C for 14 days with shaking. Culture was mixed with sterilized 50% glycerol, suspended, made 100 aliquots of 1 ml BCG solution, and then kept at -80°C. An aliquot of the stock BCG solution was inoculated in 100 ml of 7H9-ADC broth and cultured at 37°C for 10 days with shaking. BCG cells were harvested by centrifugation at 3000 rpm for 5min and suspended with 10 ml of chilled 10% glycerol.
  • BCG cells were resuspend with 5 ml of chilled 10% glycerol. This step was repeated twice. Finally, BCG cells were resuspended with 2 ml of 10% glycerol. 100 ⁇ l of BCG cell solution was taken and mixed with 2 ⁇ g of the the expression plasmid pSO-gagE in a cuvet (0.2 cm gap) for Gene-Pulser (Bio-Rad). Electroporation was done at 2500V, 25 ⁇ F and 1000 ohm. The cells were chilled on ice for 5 min, added with 150 ⁇ l of 7H9 broth and incubated at 37°C for 2 hrs. BCG cells were spread on 7H10-agar plate containing 20 ⁇ g/ml of kanamycin and incubated at 37°C for 3 weeks.
  • gagE gene was cut out by Munl-BamHl from pUC-hspK-gagE, blunted with klenow fragment and then cloned to an Smal site of pUC-wp7.5H (Ishii et al. 2002).
  • the gag E gene expression unit was cut out with HindIII and subcloned to pUC-DIs vector (Ishii et al. 2002). The resulting plasmid was named pUC-DIs-gagE.
  • CEF Embryos were taken from 8 days chicken egg (10 eggs). After removing eyes, brain and internal organs in PBS, embryos were cut to small pieces by seizer, treated with 50 ml of 0.02% EDTA-PBS in a 50 ml tube. After centrifugation at 2000 rpm for 5 min, cells were suspend in 100 ml of PBS-trypsin (0.05%) in 500 ml flask and mixed with sterilized magnetic stirrer for 30 min gently. 50 ml of supernatant was taken by decantation and poured in 10 ml of chilled FBS. The cell suspension was added with 50 ml of PBS-trypsin solution and stirred for 30 min. This step was repeated four times.
  • Recombinant vaccinia virus DIs strains construction Culture medium was removed from a CEF monolayer in 7cm plate and added with 0.4 ml of rDIs-Z ⁇ cZ virus (Ishii et al. 2002) solution (2x106 pfu in MEM-1%FBS) and shake gently at every 20 min for 1 hr. Fresh MEM-5% FBS medium (2 ml) was added and incubated overnight.
  • the pUC-DIs-gr ⁇ gE plasmid was transfected into rDIs-Z ⁇ cZ-infected CEF using Clonfectin (Clontech Co.
  • 5-bromo-4-chloro-3-indolyl-beta-D-galactoside The composition of used medium was 1.2% agar, 0.225% NaHCO3, 0.0292% L-GIn, 80 ⁇ g/ml of X-gal, 40 ⁇ g/ml of kanamycin in MEM.
  • Agar piece of colorless plaque was picked up by Pasteur pipette and put into 1 ml of MEM-1% FBS medium in eppendorf tube. After frozen and thawed, the agar piece was sonicated for 5 min and kept at -80°C or used for infection of fresh CEF. This blue-white selection step was repeated 3 or 4 times until all plaques show colorless in one plate.
  • cell and supernatant were collected, homogenized by sonication for 10 min, and used for infection of fresh CEF in 10 cm plate. After incubation at 37°C for 3 days in the complete medium, cells were harvested, washed with PBS twice and sonicated in 0.5 ml of PBS for 10 min to prepare cell lysate.
  • Recombinant DIs virus purification rDIs-GagE-infected CEF cell lysate was used for infection of new CEF monolayer in 20 of 75cm 2 flasks and cultured at 37 0 C for 3 days. After harvest, both cell and supernatant were frozen and thawed twice, sonicated for 10 min twice to homogenize cell and then applied to purification.
  • the purification procedure is as follows; (i) Put 38 ml of cultured supernatant gently on 5 ml of 36% sucrose solution per 1 tube (BECKMAN Ultra-Clear 1 x
  • CEF was cultured in 48 well cell culture plate (2.5 x 105 cell/well) a t
  • MEM- 1% FBS were used for infection of CEF (45 ⁇ l/ well). After incubation for 1 hr, 0.5 ml of fresh MEM-5% FBS medium was added and incubated for
  • 5% formalin solution in PBS
  • the cell was stained with 200 ⁇ l/well of 0.02% methylene blue (in PBS) at room temperature for 3 hrs, washed with PBS and then CPE was counted to calculate the virus titer.
  • the titer of 1 st lot was approximately 10 5 pfu/ml.
  • 1 st lot virus was infected to fresh CEF cell in 20 of 75 cm 2 flasks, cultured, purified as mentioned above. Finally the rDIs-GagE virus titer was adjusted at IxIO 8 pfu/ml.
  • Freshly isolated PBMC were added with either Concanavalin A (ConA) or 0.2 ⁇ M of pooled Gag peptides (AIDS Research and Reference Reagent Program) and were then incubated for 16 hr at 37 0 C in 5% CO2 in anti-IFN y -coated plates followed by lysing with ice-cold deionized water. After washing the plate, rabbit anti-IFN ⁇ polyclonal biotinylated detector antibody (1 ⁇ g per well; U-CyTech-BV) was added and the plates were, further, incubated for lhr at 37°C.
  • ConA Concanavalin A
  • Gag peptides AIDS Research and Reference Reagent Program
  • the plates were then washed with PBST, after which 50 ⁇ l of gold-labeled anti-biotin immunoglobulin G (GABA) solution (U-CyTech-BV) was added and incubated for lhr at 37°C. Following PBST wash, activator mix (30 ⁇ l per well; U-CyTech-BV) was added and the plates were allowed to develop for 15 min.
  • GABA gold-labeled anti-biotin immunoglobulin G
  • SFC spot-forming cells
  • FITC fluorescein isothiocyanate
  • PerCP peridinin chlorophyl protein
  • FACS fluorescence-activated cell sorter
  • the cells were washed, stained with phycoerythrin (PE) -conjugated anti-human IFN y antibody (4S.B3; BD Pharmingen), and fixed with 2% paraformaldehyde. Samples were analyzed with a FACS Callibur (Becton Dickinson), using Cell Quest software (Becton Dickinson).
  • PE phycoerythrin
  • an effective immune induction against HIV-I CRFO 1_AE using a recombinant BCG in combination with certain booster such as recombinant vaccinia virus can be attained in accordance with the invention of this application.
  • This invention would achieve effective prevention from HIV-I CEF01_AE infection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Virology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • AIDS & HIV (AREA)
  • Zoology (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Communicable Diseases (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

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

DESCRIPTION
A Method of Prime-Boost Vaccination
Technical Field
The invention of this application relates to a method of prime-boost vaccination for AIDS syndromes caused by HIV-I CRFO 1_AE strain.
Background Art
The explosive spread of human immunodeficiency virus type 1
(HIV-I) has been a serious problem in Southeast Asian countries. In Thailand, nearly 1.3 million people are infected with HIV-I, and it is claimed that there were nearly 200,000 AIDS patients by the end of year 2002. A recent report has demonstrated that CCR5-tropic CRF01_AE viruses are found in most incidence cases in the drug user cohort (Subbarao et al., 2000) and this recombinant form is now dominant in this country. So there is an urgent need to develop a prophylactic vaccine against HIV-I CRFO 1_AE that should incorporate CRF01_AE-derived antigens or epitopes. There are a few candidate vaccines at the phase III stage such as AIDSVAX B/E recombinant gpl20 (Berman et ah, 1999, Migasena et at, 2000) and recombinant canarypox virus in combination with AIDSVAX B/E. However, other vector-based vaccine has not been applied to develop AIDS vaccine against CRFO 1_AE until now.
Attenuated BCG strain of Mycobacterium bovis (hereinafter, referred to as "BCG") is one of the most popular live bacterial vaccines used for humans with quite a low risk of serious complications. This vaccine is well known to induce a strong and long-lasting T helper type 1 response, which is important to activate and maintain cytotoxic T lymphocyte (CTL). There have been many reports that demonstrate the potential immunogenicity of recombinant BCG (rBCG) vaccine targeted to HIV-I and simian immunodeficiency virus (SIV) (Honda et al., 1995, Lagranderie et at, 1997, Yasutomi et at, 1995). However, there is no candidate AIDS vaccine using recombinant BCG vector alone because of low HIV or SIV specific CTL response with usual dose for human.
Some of the inventors of this application have invented a recombinant BCG vaccine used for priming vaccination against infectious diseases, especially AIDS syndromes and filed a patent application titled "BCG Vaccine and Utilization Thereof (WO 03/097087 Al: hereinafter, referred to as "Prior Application 1"). In this Prior Application 1, use of a recombinant vaccinia virus strain DIs for boosting vaccination is also disclosed.
Disclosure of the Invention.
The invention of this application has an object of providing a novel vaccination strategy against HIV-I CRFO 1_AE by utilizing and developing the inventions of the Prior Application 1. For the object, the present inventors have found that in the case of using recombinant BCG as a priming antigen in combination with other viral vector-based vaccine as a boosting antigen, quite efficiently enhanced cellular immune response could be induced against HIV-I CRFO 1_AE whereupon the present invention has been achieved.
An invention of this application is 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-I CRFO 1_AE strain.
In said invention, it is a preferred embodiment that both of the recombinant vaccines have at least gag gene of HIV-I CRF01_AE strain. In this embodiment, it is further preferred that the gag gene encodes the amino acid sequence of SEQ ID NO: 2, and further the gag gene has the nucleotide sequence of SEQ ID NO: 1.
In said invention, it is another preferred embodiment that the gene of the recombinant BCG vaccine is modified so that a third position of each codon is substituted with G or C without a change of an amino acid.
In said invention, it is also a preferred embodiment that the recombinant vaccine for boosting step is a recombinant vaccinia virus strain DIs. In this embodiment, it is futher preferred that the boosting step with the recombinant vaccinia virus strain DIs comprises at least two trials.
In said invention, it is still further preferred embodiment that the vaccines are administered at the dose of 0.05 to 0.1 mg per subject.
Another inventions are gag gene of HIV-I CRF01_AE strain having the nucleotide sequence of SEQ ID NO: 1, and a recombinant BCG vaccine having said gag gene. Still another inventions are Gag protein of HIV-I CRFO 1_AE strain, which is a expression product of said gag gene, and an antibody specifically recognizing said Gag protein.
Terms and concepts in the present invention will be defined concretely in the description of embodiments and examples of the invention. In addition, various kinds of techniques to be used for carrying out the invention can be easily and reliably conducted by a person skilled in the art in accordance with known publications or the like, except for particular techniques cited the sources thereof. For instance, preparations of vaccine are described in Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA, 1990, and genetic engineering and molecular biological techniques are described in Sambrook and Maniatis, in Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Wiley 8s Sons, New York, N.Y., 1995, and so on.
Further, the inventions set forth above are completed by developing the inventions of said Prior Applications 1-3, and all of the Prior Applications 1-3 are incorporated by reference into the inventions of this application.
Thus, in accordance with the method of this invention, the HIV-I antigen specific immune responses can be efficiently enhanced in animal models.
Brief Description of Drawings
Fig. 1. Structure of HIV-I CRFO 1_AE Gag expression vector in BCG and western immunoblot analysis of rBCG clone. Arrow of hspδO promoter indicates direction of transcription. Km, Ori-mycobacteria, and MCS indicate kanamycin resistance gene, DNA fragment containing replication origin in mycobacteria derived from pAL5000 plasmid, and multi-cloning sites, respectively. The Gag antigen in rBCG cell lysate was fractionated on sodium dodecyl sulfate-polyacrylamide gel electrophoreisis, transferred onto nitrocellulose membrane filter and visualized with. anti-HIV-1 Gag p24 monoclonal antibody.
Fig. 2. Structure of rDIs virus expressing HIV-I CRF01_AE Gag antigen and western immunoblot analysis of rDIs-infected CEF cell lysate. W-DIs, pll, and p7.5 indicate non-recombinant DIs genome, vaccinia pl l and p7.5 promoter genes, respectively. SIV Gag antigen in rDIs-infected CEF cell lysate was analysed by the same procedure described in Fig. 1 legend.
Fig. 3. CTL activities elicited by immunization of BALB/c mice with O. lmg rBCG-GagE s.c. once for 1 month. Spleen cells were isolated and restimulated with each group of 5 serial peptides resulting in ten groups occupying ten different regions of entire HIV-I gag as the effector cells, while P815 cells were infected with recombinant vaccinia virus containing HIV-I gag and 51Cr-labelled as the target cells. Specific lysis against each peptide group was shown as number 1 to 10, while those of mice injected with rBCG/pSO246 was as immunization control.
Fig. 4. The bars indicate % specific lysis at Effector : Target 100: 1 of rBCG/ HIV-I gag immunized spleen cells against restimulation with different HIV-I Gag peptide pools occupying ten gag peptide regions.
Fig. 5. Interferon-gamma ELISpot activity in rBCG-GagE-primed and rDIs-GagE-boosted monkeys. rBCG-GagE (0.1 mg) was primed at day 0 intradermally and rDIs-GagE (107 pfu) was boosted twice at 10 and 15 weeks post-priming. After stimulation with recombinant Gag antigen of CRFO 1_AE, interferon- gamma secreting peripheral blood mononuclear cells were counted by using conventional kit.
Fig. 6. Flow cytometric analysis of IFN-gamma-producing CD8+ T cells specific for SIV Gag. PBMC from macaques were cultured in vitro with overlapping peptides and stained for intracellular IFN-gamma.
Best Mode for Carrying Out the Invention
The present invention is 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, and the character of this vaccination method is that both of the recombinant BCG vaccine for priming step and the recombinant vaccine for boosting steps have at least one gene of HIV-I CRFO 1_AE strain.
In this method, the recombinant BCG vaccine contains a recombinant BCG as an active ingredient. The recombinant BCG is a BCG strain transformed by an expression vector having at least one gene of HIV-I CRFO 1_AE strain. With regard to the BCG strain, it is possible to use widely known ones which have been used for practical vaccination against tuberculosis. With regard to the expression vector, it is possible to use a vector for mycobacteria (such as a plasmid pSO246) which has been used for the preparation of conventional recombinant BCG vaccine. The expression vector can be constructed by inserting gene(s) of HIV-I CRFO 1_AE into a cloning site of this vector. In addition, any promoter and terminator sequences derived from BCG strain (such as promoter and terminator sequences of heat shock protein (hsp) derived from BCG) and/or those derived from other mycobacterial strains are ligated to the gene(s), whereupon the gene-product(s) from HIV-I CRFOl-AE is well expressed in BCG.
A gene for insertion into BCG strain is a polynucleotide which encodes antigenic protein from any HIV-I CRF01_AE strain. To be more specific, gag precursor p55, p24 protein, env protein gpl20, gpl60 or gp41, pol protein reverse transcriptase, nef protein, tat protein, etc. which are antigenic proteins of HIV-I CRFO 1_AE. Among these, gag gene product (SEQ ID NO: 2) is more preferable. Concretely, an expression vector may be constructed by inserting the polynucleotide of SEQ ID NO: 1 into pSO246.
With regard to a method to obtain the antigenic gene, the significant sequence for that is cut out by appropriate restriction enzymes from genome gene of HIV-I CRFO 1_AE strain, or cloned plasmid cDNA. Alternatively, it may be amplified by a polymerase chain reaction (PCR) using primers of appropriate sequences using DNA or RNA derived from animal cells infected with HIV-I CRFO 1_AE strain as a template.
As an antigenic gene for the recombinant BCG, the present invention provide gag gene of HIV-I CRFO 1_AE strain, which has the nucleotide sequence of SEQ ID NO: 1. The present invention further provide a recombinant BCG vaccine holding the gag gene HIV-I CRFO 1_AE strain.
The expression vector constructed as above is introduced into BCG strain by known methods such as a calcium chloride method or an electr op oration method and expression of the polypeptide is confirmed by western blotting or by known immunological measuring method (such as ELISA) whereby the recombinant BCG of this invention can be prepared. When the recombinant BCG thus prepared is suspended in a liquid carrier which is similar to that in the case of usual BCG vaccine, a recombinant BCG vaccine can be prepared and the resulting vaccine is able to be actually used for an immune induction.
A preferred embodiment of this invention is that the inserted gene of the recombinant BCG vaccine is modified so that a third position of each codon is substituted with G or C without a change of an amino acid. The substitutions in the respective codons are shown in Table 1 in a concretive manner (the column of "optimal codon"). That is, for example, there are four codons for encoding glycine (GIy): GGT, GGC, GCA, and GGG. The GIy codon agreed with the above criteria is GGC or GGG. Therefore, the GIy codon in the amino acid sequence of some antigenic protein is GGT or GGA, the third T (thymine) or A (adenine) is substituted with C or G.
Table 1
Figure imgf000011_0001
In this invention, a preferable mode is that all positions in each codon is substituted so as to include G or C as much as possible under the conditions in which the type of an amino acid residue encoded by such a codon is not changed. Such a kind of the substitution can be applied on leucine (Lue) and arginine (Arg). That is, among the optimal codons shown in Table 1 , CTC or CTG is preferably selected as a Leu codon rather than the codon (TTG) containing two "T"s. In addition, CGC or CGG is preferably selected as an Arg codon rather than the codon (AGG) containing "A".
The codon substitution as described above is based on the following findings. That is, it is known that the BCG genome consists of DNA with a high G + C contents and the third position of the codon strongly prefers GC pair (J. Virol. 75: 9201-9209, 2001; Infect. Immun. 57: 283-288, 1989). Furthermore, from the accumulated information on BCG genes (Nucl. Acids Res. 28: 292, 2000), it is also known that the AGA codon for Arg and the TTA codon for Leu are less frequently used (0.9% and 1.6% of total codons, respectively). On the other hand, for instance, it is known that HIV-I prefers an AT pair at the third position of the codon. In other words, in the coding sequence of the HIV-I p24 gene, 9 out of 11 Arg codons use AGA and 6 out of 18 Leu codons use TTA. It is generally known that the preference of frequency in codon usage is correlated with the amount of corresponding aminoacyl tRNA in unicellular organisms (Nature 325: 728-730, 1987; MoI. Biol. Evol. 2: 13-34, 1985). It is considered that the amount of the aminoacyl tRNA for the Arg codon (AGA) and the Leu codon (TTA), which are preferred for the HIV-I p24 gene, would be quite low in the BCG cell.
Accordingly, in this preferred embodiment, the gene for insertion into recombinant BCG is designed to become a base sequence agreed with the frequency of codon usage particularly preferable for the BCG cell (i.e., the third position of the codon is G or C, and furthermore the codon contains G or C as much as possible).
For introducing a preferable base substitution corresponding to each codon into the gene, the well-known Kunkel method (Proc. Natl. Acad. Sci. USA 82: 488, 1985 and Methods in Enzymology 154: 367, 1987), well-known methods such as one using a mutation kit, a mutation-inducing type PCR method, and so on may be applied.
Regarding the second component of the prime-boost method of this invention, any booster antigen which expresses the same antigenic gene as the above recombinant BCG vaccine can be used. Recombinant viral and bacterial vectors such as adenovirus, poliovirus, influenza virus, rhinovirus, varicella virus, vaccinia virus, Salmonella and Listeria species with the same polypeptide gene as the recombinant BCG vaccine (primer vaccine). Especially, a recombinant vaccinia virus strain DIs (Prior Application 2) is a preferred booster vaccine. It is futher preferred that the boosting step with the recombinant vaccinia virus strain DIs comprises at least two trials.
Administration of prime and booster vaccines can be carried out by known methods such as injection or oral administration. Although the dose, route and schedule may depend on type (human or animal), body weight, type of the immunity to be induced, etc. of the individual to be examined, primer vaccine 0.05 to 1 mg and booster vaccine may be 105 to 1010 plaque forming units for example. The time interval between two inoculations of vaccine may be 2 to 12 months.
The present invention further provides gag gene of HIV-I CRFO 1_AE strain that has the nucleotide sequence of SEQ ID NO: 1, and a recombinant BCG vaccine holding the gag gene, which has been deposited under Budapest treaty at IPOD as a deposit number BP-OOOO.
The gag gene of this invention is different from the known gag gene as shown in Fig. 7, and therefore it is novel gene. Consequently, the novel gag gene of this invention has utilities as follows.
The gag gene of this invention can be used as an insert for constructing the recombinant BCG for priming vaccine, and also for constructing the boost vaccine such as the recombinant vaccinia DIs. One of ordinary skill in the art may obtain the gag gene from the deposited BCG by, for example, using appropriate restriction enzymes or PCR method. As to the PCR method, the set of primers as described in Example 1, i.e. those of having SEQ ID NO.:3 and 4, may be used. The gag gene of this invention also can be utilized for diagnosing an
AIDS patient as to whether the patient would be infected with the HIV-I
CRFO 1_AE strain. The diagnosis may be performed, for example, by directly sequencing the gag gene isolated from the patient and comparing the sequence with that of SEQ ID NO: 1 of this invention. Alternatively, PCR method of using a set of specific primers to the SEQ ID NO: 1 may be employed for the diagnosis. That is, if the patient is infected with the HIV-I
CRFO 1_AE strain, the unique gag gene is amplified with the PCR method. Further, the diagnosis can be performed with DNA microarray system that comprises probes specific to the sequence of SEQ ID NO: 1.
The gag gene of this invention can be further used for producing Gag protein of HIV-I CRFO 1_AE strain, which is also an invention provided by the present invention. The Gag protein of this invention may be useful as a target protein for developing pharmaceuticals against AIDS, especially the HIV-I CRFO 1_AE strain-infectious disease. The Gag protein may be also used for developing a diagnostic agent for the HIV-I CRFO 1_AE strain-infectious disease. For example, using the Gag protein as an immunogen would make an antibody against the HIV-I CRF01_AE strain.
The Gag protein of this invention, Le., polypeptide having the amino acid sequence of SEQ ID NO: 2, can be prepared by known methods such as chemical synthesis of peptide based upon the amino acid sequence provided by this invention, or a recombinant DNA technique using the gag gene provided by this invention. For example, in case the Gag protein is prepared by means of a recombinant DNA technique, by expressing the gag gene in a suitable host-vector system, it is possible to obtain the Gag protein in a large quantity in Escherichia coli, Bacillus subtilis, yeast, insect cells, animal cells, etc. The antibody of this invention is polyclonal antibody or monoclonal antibody, and it includes hole molecule binding to epitope of the Gag protein, Fab* F(ab')2% Fv fragment. The polyclonal antibody may be obtained from serum of an animal immunized with the Gag protein or its partial peptide. The monoclonal antibody can be obtained according to the know method as described in "Monoclonal Antibody" James W. Goding, third edition, Academic Press, 1996.
The invention of this application will now be illustrated as hereunder in more detail and specifically by the following Examples even though the invention of this application is not limited by the Examples.
Examples
Construction of expression vectors
A DNA fragment encoding hspδO gene of BCG (Thole et al. 1987) was cloned into Smal-SaU. sites of pUC18 (pUC-hsp60). A synthetic DNA fragment which corresponds to multi-cloning site and terminator region of hsp60 gene was cloned into Munl-Kpnl sites of pUC-hsp60 and then inserted Kpnl linker at EcoRl site giving rise to pUC-hspK vector. The gag gene from HIV-I CRFO 1_AE clinical isolate M33 was amplified by PCR from PBMC of a Thai patient. Used primers were as follows: forward primer:
5'-ATATATCAATTGATCTAGCGGAGGCTAGAAGGAGAGAG-S' (SEQ ID NO:3) reverse primer: 5'-ATATAATGGATCCCTAATACTGTATCATCTGCTCCTGTAT-S' (SEQ ID NO:4) Munl-BamHl cut PCR product was cloned into the same sites of pUC-hspK vector, giving rise to pUC-hsp-gagE. This plasmid was cut with Kpnl and a small fragment was subcloned to pSO246 (Matsumoto et al. 1994) to generate pSO-gagE (Fig. IA).
Transformation of BCG Tokyo strain
Seed lot of BCG Tokyo substrain was inoculated into 50 ml of 7H9-ADC broth and cultured at 37°C for 14 days with shaking. Culture was mixed with sterilized 50% glycerol, suspended, made 100 aliquots of 1 ml BCG solution, and then kept at -80°C. An aliquot of the stock BCG solution was inoculated in 100 ml of 7H9-ADC broth and cultured at 37°C for 10 days with shaking. BCG cells were harvested by centrifugation at 3000 rpm for 5min and suspended with 10 ml of chilled 10% glycerol. After centrifugation at 2500 rpm for 5 min, BCG cells were resuspend with 5 ml of chilled 10% glycerol. This step was repeated twice. Finally, BCG cells were resuspended with 2 ml of 10% glycerol. 100 μl of BCG cell solution was taken and mixed with 2 μg of the the expression plasmid pSO-gagE in a cuvet (0.2 cm gap) for Gene-Pulser (Bio-Rad). Electroporation was done at 2500V, 25 μF and 1000 ohm. The cells were chilled on ice for 5 min, added with 150 μl of 7H9 broth and incubated at 37°C for 2 hrs. BCG cells were spread on 7H10-agar plate containing 20 μg/ml of kanamycin and incubated at 37°C for 3 weeks.
Expression of GagE antigen in BCG Transformants of BCG were picked up, cultured on a new 7H10-agar plate containing 20 μg/ml of kanamycin for 2 weeks and then grown in 30 ml of 7H9-ADC broth containing 20 μg/ml of kanamycin for 2 weeks. Two ml of culture was harvested, washed with 0.5 ml of Tris buffered saline (TBS) twice and then sonicated in 200 μl of TBS. Ten μl of cell sonicate was applied to sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Multi-Gel 4/20 (Daiichi Chemical Co., Japan). Fractionated proteins were electroblotted onto a nitrocellulose membrane filter, reacted with corresponding monoclonal antibodyies and visualized with a substrate (3,3'-diarninobenzydine) of peroxidase. Finally rBCG clones which expressed GagE antigen was successfully obtained and named rBCG-GagE (Fig. IB).
Construction of transfer vector for gagE gene. On the other hand, we obtained rDIs viruses that produce CRFO 1_AE Gag antigen in infected chick embryo fibroblast (CEF). Briefly, the gag E gene was cut out by Munl-BamHl from pUC-hspK-gagE, blunted with klenow fragment and then cloned to an Smal site of pUC-wp7.5H (Ishii et al. 2002). The gag E gene expression unit was cut out with HindIII and subcloned to pUC-DIs vector (Ishii et al. 2002). The resulting plasmid was named pUC-DIs-gagE.
Preparation of CEF Embryos were taken from 8 days chicken egg (10 eggs). After removing eyes, brain and internal organs in PBS, embryos were cut to small pieces by seizer, treated with 50 ml of 0.02% EDTA-PBS in a 50 ml tube. After centrifugation at 2000 rpm for 5 min, cells were suspend in 100 ml of PBS-trypsin (0.05%) in 500 ml flask and mixed with sterilized magnetic stirrer for 30 min gently. 50 ml of supernatant was taken by decantation and poured in 10 ml of chilled FBS. The cell suspension was added with 50 ml of PBS-trypsin solution and stirred for 30 min. This step was repeated four times. Total 300 ml of cell suspension was passed through sterilized mesh to remove cell debris, centrifuged at 2000 rpm for 5 min, and resuspended in 500ml of Eagle's modified essential medium supplemented with 5% FBS (MEM-5% FBS). This suspension, which had about 1x10« cells per ml, was poured into plastic plate (10 ml for large, 3 ml for small one) and incubated at 37°C for 3 days to obtain monolayer of CEF.
Recombinant vaccinia virus DIs strains construction Culture medium was removed from a CEF monolayer in 7cm plate and added with 0.4 ml of rDIs-ZαcZ virus (Ishii et al. 2002) solution (2x106 pfu in MEM-1%FBS) and shake gently at every 20 min for 1 hr. Fresh MEM-5% FBS medium (2 ml) was added and incubated overnight. The pUC-DIs-grαgE plasmid was transfected into rDIs-ZαcZ-infected CEF using Clonfectin (Clontech Co. Ltd) to cause homologous recombination of lacZ gene to gag E gene at a major deletion site of parent vaccinia virus DIs strain (Fig. 2A). After incubation for 3 days, both cell and supernatant were collected, repeated freezing and thawing twice and sonicated to be homogenize cell. Serially diluted virus solution was used for infection to newly preparated CEF and incubated for 3 days in MEM-5% FBS medium. After removing medium, colorless plaques were selected on MEM medium-agar plate containing 0.004%
5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). The composition of used medium was 1.2% agar, 0.225% NaHCO3, 0.0292% L-GIn, 80 μg/ml of X-gal, 40 μg/ml of kanamycin in MEM. Agar piece of colorless plaque was picked up by Pasteur pipette and put into 1 ml of MEM-1% FBS medium in eppendorf tube. After frozen and thawed, the agar piece was sonicated for 5 min and kept at -80°C or used for infection of fresh CEF. This blue-white selection step was repeated 3 or 4 times until all plaques show colorless in one plate. From one 7cm diameter plate, cell and supernatant were collected, homogenized by sonication for 10 min, and used for infection of fresh CEF in 10 cm plate. After incubation at 37°C for 3 days in the complete medium, cells were harvested, washed with PBS twice and sonicated in 0.5 ml of PBS for 10 min to prepare cell lysate.
Expression of Gag E antigen in rDIs-infected CEF
Ten μl of cell lysate was applied to SDS-PAGE using Multi-Gel 4/20
(Daiichi Chemical Co., Japan). Fractionated proteins were electroblotted onto a nitrocellulose membrane filter, reacted with anti-HIV gag V107 monoclonal antibody (Matsuo et al. 1994). After reacting with peroxidase-conjugated anti-mouse IgG as a secondary antibody, the reactive protein was visualized with a substrate (3,3'-diaminobenzydine) of peroxidase. From the western blot analyses, rDIs clones which expressed gag E gene in CEF were obtained and named as rDIs-GagE (Fig. 2B) .
Recombinant DIs virus purification rDIs-GagE-infected CEF cell lysate was used for infection of new CEF monolayer in 20 of 75cm2 flasks and cultured at 370C for 3 days. After harvest, both cell and supernatant were frozen and thawed twice, sonicated for 10 min twice to homogenize cell and then applied to purification. The purification procedure is as follows; (i) Put 38 ml of cultured supernatant gently on 5 ml of 36% sucrose solution per 1 tube (BECKMAN Ultra-Clear 1 x
3.5 in. 25 x 89 mm) and centrifuge at 14000 rpm for 80 min. (ii) Resuspend precipitate with 5ml, 25 ml and then 8 ml of PBS (three times), put on 5 ml of 36% sucrose solution in centrifugation tube gently, and then centrifuge at
17000 rpm for 30 min. (iii) Wash precipitate gently with PBS, suspend in 100 ml of PBS completely and divide into crio-tube (1 ml per tube), and then keep them at -800C (l»t lot).
Recombinant DIs virus titration
CEF was cultured in 48 well cell culture plate (2.5 x 105 cell/well) at
370C in CO2 incubator for 3 days. 10-fold serial dilutions of virus in
MEM- 1% FBS were used for infection of CEF (45 μl/ well). After incubation for 1 hr, 0.5 ml of fresh MEM-5% FBS medium was added and incubated for
3 days. After removing the cultured medium, cell was fixed in 200 μl/well of
5% formalin solution (in PBS) at room temperature for overnight. The cell was stained with 200 μl/well of 0.02% methylene blue (in PBS) at room temperature for 3 hrs, washed with PBS and then CPE was counted to calculate the virus titer. The titer of 1st lot was approximately 105 pfu/ml. To obtain higher titer of rDIs-GagE, 1st lot virus was infected to fresh CEF cell in 20 of 75 cm2 flasks, cultured, purified as mentioned above. Finally the rDIs-GagE virus titer was adjusted at IxIO8 pfu/ml.
Cellular immune response in mice
Inoculation of rBCG-GagE to BALB /c elicited peptide-antigen-specific CTL in the immunized animals (Fig. 3 and 4). rDIs-GagE construct also induced quite a high CTL response in mice and was clearly demonstrated to be highly immunogenic in spite of its replication incompetence. Consequently, we decided to evaluate these candidate vaccines for prime-boost regimen in cynomolgus monkey.
Enhanced cellular immune response by the prime and boost vaccination in monkey rBCG-GagE (0.1 mg, intradermal^) -priming and rDIs-GagE (107 pfu, intradermally) -boosting regimen, both of which were practical dose and route in humans, was evaluated for enhancing cellular immune response in cynomolgus monkey by interferon gamma-ELISpot assay. As shown in Fig. 5, two shots of rDIs-boosting was quite efficiently enhance HIV-I Gag-specific ELISpot response. Virus-specific IFN y ELISPOT assay was performed. In brief, ninety- six- well flat-bottomed plates (U-CyTech-BV, Utrecht, Netherlands) were coated with anti-IFN y mAb MD- 1 (U-CyTech-BV) overnight at 4°C. The plates were then washed with PBS containing 0.05% Tween 20 (PBST) and blocked with PBS containing 2% bovine serum albumin (PBSA) for lhr at 37°C. PBSA was discarded from the plates. Freshly isolated PBMC were added with either Concanavalin A (ConA) or 0.2 μM of pooled Gag peptides (AIDS Research and Reference Reagent Program) and were then incubated for 16 hr at 370C in 5% CO2 in anti-IFN y -coated plates followed by lysing with ice-cold deionized water. After washing the plate, rabbit anti-IFN γ polyclonal biotinylated detector antibody (1 μg per well; U-CyTech-BV) was added and the plates were, further, incubated for lhr at 37°C. The plates were then washed with PBST, after which 50 μl of gold-labeled anti-biotin immunoglobulin G (GABA) solution (U-CyTech-BV) was added and incubated for lhr at 37°C. Following PBST wash, activator mix (30 μl per well; U-CyTech-BV) was added and the plates were allowed to develop for 15 min.
Wells were imaged and spot-forming cells (SFC) were counted using the KS ELISPOT compact system (Carl Zeiss, Germany). A SFC was defined as a large black spot with a fuzzy border. To determine significance levels, a baseline for each peptide was established using the average and standard deviation of the number of SFCs for each peptide. A threshold significance value corresponding to this average plus two standard deviations was then determined. A response was considered positive if the number of SFCs exceeded the threshold significance level of the sample with no peptide. In addition, the intracellular cytokine staining assay demonstrated the enhancement of CD8+ CTL activation in the immune monkeys (Fig. 6). These data suggest that efficient positive cellular immunity could be induced and enhanced by this prime-boost regimen. To detection intracellular IFND by flow cytometry, freshly isolated PBMC (5 X 105 to I X lO6 cells) were suspended in R- 10 medium and incubated with antigen for 16 hr at 370C with 5% CC-2. During the final 6-8 hr, Brefeldin A (Sigma Chemical Co., St. Louis, MO) was added at 10 μg/tnl. Antibody to CD28 (lμg/ml, BD Pharmingen, San Diego, CA) was also added during the incubation as a co-stimulator molecule. After stimulation, the cells were washed and stained with fluorescein isothiocyanate (FITC) -conjugated anti-CD3 (FN18; Biosource, Camarillo, CA) and peridinin chlorophyl protein (PerCP)-conjugated anti-CD8 antibodies (Leu-2a; Becton Dickinson). The cells were then sequentially incubated with fluorescence-activated cell sorter (FACS) Lysing Solution (Becton Dickinson Biosciences, San Jose, CA) for 10 min and FACS permeabilizing solution (Becton Dickinson) for another 10 min. The cells were washed, stained with phycoerythrin (PE) -conjugated anti-human IFN y antibody (4S.B3; BD Pharmingen), and fixed with 2% paraformaldehyde. Samples were analyzed with a FACS Callibur (Becton Dickinson), using Cell Quest software (Becton Dickinson).
Industrial Applicability
As fully illustrated hereinabove, an effective immune induction against HIV-I CRFO 1_AE using a recombinant BCG in combination with certain booster such as recombinant vaccinia virus can be attained in accordance with the invention of this application. This invention would achieve effective prevention from HIV-I CEF01_AE infection.

Claims

1. 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-I CRFO 1_AE strain.
2. The method according to claim 1, wherein both of the recombinant vaccines have at least gag gene of HIV-I CRPO 1_AE strain.
3. The method according to claim 2, wherein the gag gene encodes Gag protein having the amino acid sequence of SEQ ID NO: 2.
4. The method according to claim 3, wherein the gag gene has the nucleotide sequence of SEQ ID NO: 1.
5. The method according to any one of claims 1 to 4, wherein the gene of the recombinant BCG vaccine is modified so that a third position of each codon is substituted with G or C without a change of an amino acid.
6. The method according to claim 1, wherein the recombinant vaccine for boosting step is a recombinant vaccinia virus strain DIs.
7. The method according to claim 6, wherein the boosting step with the recombinant vaccinia virus strain DIs comprises at least two trials.
8. The method according to any one of claims 1 to 7, wherein the vaccines are administered at the dose of 0.05 to 0.1 mg per subject.
9. gag gene of HIV-I CRF01_AE strain having the nucleotide sequence of SEQ ID NO: 1.
10. A recombinant BCG vaccine having the gag gene of claim 9.
11. Gag protein of HIV-I CRF01_AE strain, which is an expression product of the gag gene of claim 9.
12. An antibody specifically recognizing the Gag protein of claim 11.
PCT/JP2005/022221 2004-11-25 2005-11-25 A method of prime-boost vaccination WO2006057454A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004-341283 2004-11-25
JP2004341283A JP2006149234A (en) 2004-11-25 2004-11-25 Prime-boost vaccination method

Publications (1)

Publication Number Publication Date
WO2006057454A1 true WO2006057454A1 (en) 2006-06-01

Family

ID=36498175

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/022221 WO2006057454A1 (en) 2004-11-25 2005-11-25 A method of prime-boost vaccination

Country Status (3)

Country Link
JP (1) JP2006149234A (en)
CN (1) CN101107359A (en)
WO (1) WO2006057454A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020025642A1 (en) 2018-08-03 2020-02-06 Ludwig Institute For Cancer Research Ltd. Viral vectors encoding cancer/testis antigens for use in a method of prevention or treatment of cancer

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130302367A1 (en) 2010-10-22 2013-11-14 Hisatoshi Shida Virus vector for prime/boost vaccines, which comprises vaccinia virus vector and sendai virus vector
JP2014512353A (en) * 2011-03-31 2014-05-22 アムリタ セラピューティクス リミテッド Antiviral composition
CA2884859A1 (en) 2012-09-12 2014-03-20 Duke University Hiv-1 antibody evolution immunogens

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003009874A (en) * 2001-06-25 2003-01-14 National Institute Of Infectious Diseases Dna encoding infectious hiv genome
WO2003097087A1 (en) * 2002-05-20 2003-11-27 Japan Science And Technology Agency Bcg vaccine and utilization thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003009874A (en) * 2001-06-25 2003-01-14 National Institute Of Infectious Diseases Dna encoding infectious hiv genome
WO2003097087A1 (en) * 2002-05-20 2003-11-27 Japan Science And Technology Agency Bcg vaccine and utilization thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WEI M. ET AL.: "Simple subtyping assay for human immunodeficiency virus type 1 subtypes B, C, CRF01-AE, CRF07-BC, and CRF08-BC.", J. CLIN. MICROBIOL., vol. 42, no. 9, September 2004 (2004-09-01), pages 4261 - 4267, XP002998793 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020025642A1 (en) 2018-08-03 2020-02-06 Ludwig Institute For Cancer Research Ltd. Viral vectors encoding cancer/testis antigens for use in a method of prevention or treatment of cancer

Also Published As

Publication number Publication date
CN101107359A (en) 2008-01-16
JP2006149234A (en) 2006-06-15

Similar Documents

Publication Publication Date Title
Yasutomi et al. A vaccine-elicited, single viral epitope-specific cytotoxic T lymphocyte response does not protect against intravenous, cell-free simian immunodeficiency virus challenge
JP3967374B2 (en) Synchronous in vivo gene expression
AU729231B2 (en) Synthetic HIV genes
US8183352B2 (en) Attenuated vif DNA immunization cassettes for genetic vaccines
Lai et al. GM-CSF DNA: an adjuvant for higher avidity IgG, rectal IgA, and increased protection against the acute phase of a SHIV-89.6 P challenge by a DNA/MVA immunodeficiency virus vaccine
Montefiori et al. Induction of neutralizing antibodies and gag-specific cellular immune responses to an R5 primary isolate of human immunodeficiency virus type 1 in rhesus macaques
KR20000070865A (en) Synthetic HIV gag genes
EP2205736A1 (en) Hiv preventive vaccine based on hiv specific antibodies
Williamson et al. Justification for the inclusion of Gag in HIV vaccine candidates
Billaut-Mulot et al. Interleukin-18 modulates immune responses induced by HIV-1 Nef DNA prime/protein boost vaccine
Osterhaus et al. Isolation and partial characterization of a lentivirus from talapoin monkeys (Myopithecus talapoin)
GARDNER Vaccination against SIV infection and disease
WO2006057454A1 (en) A method of prime-boost vaccination
Cherpelis et al. DNA-immunization with a V2 deleted HIV-1 envelope elicits protective antibodies in macaques
Someya et al. A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity
EP2021356B1 (en) Hiv vaccine
Girard et al. Candidate vaccines for HIV
US20020127238A1 (en) HIV-1 vaccines and screening methods therefor
Chugh et al. Induction of broad-based immune response against HIV-1 subtype C gag DNA vaccine in mice
US20090270488A1 (en) Process for the Selection of HIV-1 Subtype C Isolates, Selected HIV-1 Subtype Isolates, Their Genes and Modifications and Derivatives Thereof
Evans et al. HIV-1 epitope expression on a chimaeric poliovirus vaccine vector
Barrett et al. Large-scale production, purification and immunological analysis of vaccinia recombinant derived HIV-1 gp160
Gardner et al. Genetic Immunization for Lentiviral Immunodeficiency Virus Infection and Disease.
Adams et al. Towards an AIDS vaccine-recombinant Ty: HIV virus-like particles
Dalgleish et al. Potential of the anti-idiotype approach for the development of an anti-HIV vaccine

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 200580040572.1

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05811667

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 5811667

Country of ref document: EP