WO2016062226A1 - 复制型痘苗病毒载体艾滋病疫苗 - Google Patents

复制型痘苗病毒载体艾滋病疫苗 Download PDF

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WO2016062226A1
WO2016062226A1 PCT/CN2015/092170 CN2015092170W WO2016062226A1 WO 2016062226 A1 WO2016062226 A1 WO 2016062226A1 CN 2015092170 W CN2015092170 W CN 2015092170W WO 2016062226 A1 WO2016062226 A1 WO 2016062226A1
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vaccinia virus
hiv
recombinant
polynucleotide
seq
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邵一鸣
张其程
刘颖
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中国疾病预防控制中心性病艾滋病预防控制中心
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Priority to EP15852404.1A priority Critical patent/EP3211073A4/en
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Definitions

  • the present invention relates to the field of medical biotechnology, and in particular to a replicative vaccinia virus AIDS vaccine capable of inducing high levels of humoral and cellular immune responses against human immunodeficiency virus and allowing effective control of serious adverse reactions that may be caused by the vaccine.
  • the vaccinia virus vector is one of the most intensive and extensive viral vectors and can be used as a vector for constructing vaccines.
  • the non-replicating vaccinia virus vector has high safety, but the immunogenicity is relatively weak, especially the immune response induced in the human body is significantly weaker than the immune response in the monkey.
  • the replicating vaccinia virus vector is highly immunogenic and can induce long-lasting immunity, but has potential safety problems such as serious adverse reactions.
  • the invention provides a recombinant replication vaccinia virus comprising a first polynucleotide encoding HSV-TK and a second polynucleotide encoding an HIV antigen in the TK region.
  • the recombinant replication vaccinia virus of the present invention is a vaccinia virus Tiantan strain.
  • the first polynucleotide of the recombinant replication vaccinia virus of the invention comprises the nucleotide sequence set forth in SEQ ID NO:3.
  • the first polynucleotide of the recombinant replication vaccinia virus of the invention comprises at least 80%, at least 85%, at least 90 of the nucleotide sequence set forth in SEQ ID NO:3 a nucleotide sequence of %, at least 95%, or at least 99% identity, and the first polynucleotide encodes a TK from HSV-1.
  • the second polynucleotide of the recombinant replication vaccinia virus of the present invention encodes gp145 2M derived from the major HIV strain of China, 97CN001, and comprises the SEQ ID NO: 1 Nucleotide sequence.
  • the present invention The second polynucleotide in the recombinant recombinant vaccinia virus encodes gp145 2M derived from the major HIV strain of China, 97CN001, or an immunogenic fragment thereof.
  • the second polynucleotide of the recombinant replication vaccinia virus of the invention comprises at least 80%, at least 85%, at least 90 of the nucleotide sequence set forth in SEQ ID NO: a nucleotide sequence of %, at least 95%, or at least 99% identity, and the second polynucleotide encodes gp145 2M from HIV-1.
  • the recombinant replication vaccinia virus of the invention further comprises a third polynucleotide encoding an additional HIV antigen.
  • the third polynucleotide is inserted into the HA region of a replicating vaccinia virus.
  • the third polynucleotide encodes a gag derived from the major HIV strain of China, 97CN001, and comprises the nucleotide sequence set forth in SEQ ID NO: 2.
  • the third polynucleotide encodes a gag or an immunogenic fragment thereof derived from the major HIV strain of China, 97CN001.
  • the third polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the nucleotide sequence set forth in SEQ ID NO: An identical nucleotide sequence, and the third polynucleotide encodes a gag from HIV-1.
  • the present invention provides a recombinant replicating vaccinia virus which is a Tiantan strain vaccinia virus comprising a gene encoding gp145 2M from a major HIV strain of China Tianxin strain 97CN001 inserted into the TK region of the Tiantan strain vaccinia virus. And a gene encoding HSV-TK, and a gene encoding gag of 97CN001 inserted into the HA region of the vaccinia virus of the Tiantan strain.
  • the invention further provides a live vector HIV vaccine composition
  • a live vector HIV vaccine composition comprising the recombinant replication vaccinia virus of the invention of the first aspect above and a pharmaceutically acceptable carrier and/or adjuvant.
  • the present invention also relates to a method of preventing and/or treating HIV infection in a subject comprising administering to the subject a live vector HIV vaccine composition comprising the recombinant replicative vaccinia virus of the present invention of the above first aspect.
  • the present invention also relates to the use of the recombinant replicative vaccinia virus of the present invention of the above first aspect for the preparation of a live vector HIV vaccine composition for preventing and/or treating HIV infection in a subject.
  • the subject is an immunosuppressed patient, eg, the subject has a congenital immune system deficiency, HIV infection, or is undergoing immunosuppressive therapy.
  • the invention also provides an AIDS immunoassay kit comprising a plurality of components and instructions for indicating an immunization procedure, wherein one component is a live carrier vaccine composition of the invention.
  • Figure 1 In vitro inhibition of recombinant vaccinia virus by GCV.
  • Figure 2 Inhibition of TT-TK and TT-EnvTK replication by mouse GC in mouse brain.
  • Figure 3 Inhibition of TT-TK replication in mouse ovaries by GCV.
  • Figure 4 Induction of immune response by TT-EnvTK in mice.
  • A cellular immunity
  • B humoral immunity.
  • FIG. 1 Immune interference between multiple HIV antigens.
  • the recombinant vaccinia virus TT-TK was deposited on October 20, 2014 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee, deposit number CGMCC No. 9810;
  • the recombinant vaccinia virus TT-EnvTK was deposited on October 20, 2014 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee, deposit number CGMCC No. 9808;
  • the recombinant vaccinia virus TT-gag was deposited on October 20, 2014 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, under the accession number CGMCC No. 9809.
  • CGMCC General Microbiology Center
  • SEQ ID NO: 1 Gp145 2M gene sequence from HIV-1;
  • SEQ ID NO: 2 Gag gene sequence from HIV-1;
  • SEQ ID NO: 3 HSV-TK gene sequence from HSV-1;
  • SEQ ID NO:4 Plasmid pSC65-DR-GN sequence.
  • AIDS Acquired Immunodeficiency Syndrome
  • HAV human immunodeficiency virus
  • the AIDS epidemic has had a tremendous impact on the social and economic development of the world.
  • HIV infection has caused a shortened life expectancy, reduced labor, and food shortages, which have caused economic and social development to fall back for 20 years.
  • HIV in China's epidemic has experienced three stages: the introduction period (1985-1988), the dissemination period (1989-1994), and the growth period (1995-present).
  • AIDS vaccine must be able to induce the body to produce HIV-specific CD8+ cytotoxic T lymphocyte response (CTL) and neutralizing antibody responses.
  • CTL cytotoxic T lymphocyte response
  • Alternative vaccine candidates include the following: traditional vaccines (inactivated vaccines and live attenuated vaccines), synthetic peptide and protein subunit vaccines, DNA vaccines, and live vector vaccines.
  • live vector vaccines Compared with other types of vaccines, the advantages of live vector vaccines are: (1) active infection of target tissues or cells, increasing the efficiency of entry of foreign genes into cells; (2) the carrier itself has an adjuvant effect and can induce cytokines And the production of chemokines; (3) most can induce long-term immune response.
  • the research on live vector vaccine with vaccinia virus as the carrier is the most in-depth and extensive, but most of the current clinical trials are non-replicating vectors, such as MVA, NYVAC and ALVAC.
  • non-replicating vector is relatively weakly immunogenic, and that the immune response induced by the same vaccine in humans is significantly weaker than that in monkeys. This may be related to its non-replicating nature.
  • the non-replicating vector vaccine infects the host cell, it can only perform one cycle of antigen expression, processing and presentation, unable to produce an infectious virus and start a new round of infection, so its stimulation of the immune system is limited. Short-lived.
  • the research focus of live carrier vaccines in the world has shifted from non-replicating to replicating, in order to fully utilize the characteristics of live vectors that can be replicated, and to better stimulate the body to produce immune responses.
  • the replicating vaccinia virus vector is highly immunogenic and can induce long-lasting immunity, but has potential safety problems such as serious adverse reactions. Especially for individuals who have suppressed the immune system due to defects in the innate immune system, immunosuppressive therapy or HIV infection, the replicative vaccinia virus vector vaccine may cause serious adverse reactions [1, 2] .
  • the suicide gene system is widely used in the treatment of diseases such as cancer in recent years.
  • the common suicide gene/drug combination is: Cytosine deaminase (CD)/5-fluorocytosine [3] ] , Nitroreductase/CB 1954 [4] , Thymidine phosphorylase (TP)/5' deoxy-5-fluorouridine (5'-DFUR) [5] ], purine nucleoside phosphorylase (purine nucleoside phosphorylase, PNP) / 6 methyl purine 2 'deoxynucleoside (6-methylpurine-2'-deoxyriboside , MeP-dR) [6] and herpes simplex virus thymidine kinase ( Herpes simplex virus thymidine kinase, HSV-TK) / Ganciclovir (GCV) [7] .
  • HSV-TK/GCV is the most studied suicide gene system.
  • GCV activity depends on the TK gene of HSV.
  • HSV TK can phosphorylate GCV to form its monophosphate form (GCV-MP) [8] , and then the cell kinase further phosphorylates GCV-MP to form diphosphate ( GCV-DP) and the toxic triphosphate form (GCV-TP).
  • GCV-TP is a DNA polymerase inhibitor that inhibits DNA replication by competitively inhibiting the binding of deoxyguanosine triphosphate to DNA polymerase or insertion into the nascent strand, resulting in the termination of new chain elongation and eventual cell death [ 9] . Since GCV cannot be directly phosphorylated by mammalian cellular kinases, prodrugs are only selectively toxic to cells expressing HSV-TK with few adverse events.
  • HSV-TK/GCV suicide gene system is used to treat tumors, wherein the role of the HSV-TK/GCV suicide gene system is to directly kill target cells, rather than to ensure the use of live virus vectors. Safety and control of vaccine vector-related adverse effects that may occur.
  • the current HSV-TK/GCV suicide gene system is mainly used for adenoviral vectors and adeno-associated virus vectors, HSV-TK/GCV systems and vaccinia virus vectors, especially vaccinia virus Tiantan strains as replicative vaccinia virus vectors. Compatibility has not been confirmed.
  • the TK gene of vaccinia virus has only ⁇ 16% homology with HSV-TK, and ganciclovir does not seem to inhibit vaccinia virus (EC50>300 ⁇ M) [10] .
  • the present inventors introduced the HSV-TK gene into a replication-type vaccinia virus vector, thereby providing a safe and controllable replication-type vaccinia virus vector AIDS vaccine.
  • the present inventors confirmed that the introduction of the HSV-TK gene into a replicative vaccinia virus AIDS vaccine does not affect replicative vaccinia Under the premise of immunogenicity of virulence vector, it can effectively control the serious adverse reactions that may be caused by replicating vaccinia virus vector, improve the safety of vaccine, and solve the safety problem of replicating vaccinia virus vector.
  • the replicative vaccinia virus vector AIDS vaccine of the present invention is particularly suitable for immunosuppressed patients, such as subjects suffering from a defect in the innate immune system, HIV infection, or undergoing immunosuppressive therapy.
  • HIV-specific CD8+ T cell killing plays an important role in controlling HIV replication. Gag-specific CTL is beneficial for the control of viral replication in infected individuals. Env-induced neutralizing antibodies can neutralize HIV, and V1V2 binding antibody titers are positively correlated with vaccine immunoprotection. Therefore, AIDS vaccines should select multiple antigens to induce different types of immune responses. Moreover, the present inventors have found that the HIV-1 gag and gp145 2M genes are respectively introduced into the HA region and the TK region of the replication vaccinia virus vector, and the compatibility between different antigens is also avoided under the premise of ensuring that the vaccine expresses multiple antigens. interference.
  • the invention provides a recombinant replication vaccinia virus comprising a first polynucleotide encoding HSV-TK and a second polynucleotide encoding an HIV antigen in the TK region.
  • the recombinant replication vaccinia virus of the present invention is a vaccinia virus Tiantan strain.
  • the first polynucleotide of the recombinant replication vaccinia virus of the invention comprises the nucleotide sequence set forth in SEQ ID NO:3.
  • the first polynucleotide of the recombinant replication vaccinia virus of the invention comprises at least 80%, at least 85%, at least 90 of the nucleotide sequence set forth in SEQ ID NO:3 a nucleotide sequence of %, at least 95%, or at least 99% identity, and the first polynucleotide encodes a TK from HSV-1.
  • the second polynucleotide of the recombinant replication vaccinia virus of the present invention encodes gp145 2M derived from the major HIV strain of China, 97CN001, and comprises the SEQ ID NO: 1 Nucleotide sequence.
  • the second polynucleotide of the recombinant replication vaccinia virus of the invention encodes gp145 2M or an immunogenic fragment thereof derived from the major HIV strain 97CN001 of China.
  • the second polynucleotide of the recombinant replication vaccinia virus of the invention comprises at least 80%, at least 85%, at least 90 of the nucleotide sequence set forth in SEQ ID NO: a nucleotide sequence of %, at least 95%, or at least 99% identity, and the second polynucleotide encoding is from HIV-1 gp145 2M.
  • the recombinant replication vaccinia virus of the invention further comprises a third polynucleotide encoding an additional HIV antigen.
  • the third polynucleotide is inserted into the HA region of a replicating vaccinia virus.
  • the third polynucleotide encodes a gag derived from the major HIV strain of China, 97CN001, and comprises the nucleotide sequence set forth in SEQ ID NO: 2.
  • the third polynucleotide encodes a gag or an immunogenic fragment thereof derived from the major HIV strain of China, 97CN001.
  • the third polynucleotide comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the nucleotide sequence set forth in SEQ ID NO: An identical nucleotide sequence, and the third polynucleotide encodes a gag from HIV-1.
  • the present invention provides a recombinant replicating vaccinia virus which is a Tiantan strain vaccinia virus comprising a gene encoding gp145 2M from a major HIV strain of China Tianxin strain 97CN001 inserted into the TK region of the Tiantan strain vaccinia virus. And a gene encoding HSV-TK, and a gene encoding gag of 97CN001 inserted into the HA region of the vaccinia virus of the Tiantan strain.
  • the invention further provides a live vector HIV vaccine composition
  • a live vector HIV vaccine composition comprising the recombinant replication vaccinia virus of the invention of the first aspect above and a pharmaceutically acceptable carrier and/or adjuvant.
  • the present invention also relates to a method of preventing and/or treating HIV infection in a subject comprising administering to the subject a live vector HIV vaccine composition comprising the recombinant replicative vaccinia virus of the present invention of the above first aspect.
  • the present invention also relates to the use of the recombinant replicative vaccinia virus of the present invention of the above first aspect for the preparation of a live vector HIV vaccine composition for preventing and/or treating HIV infection in a subject.
  • the subject is an immunosuppressed patient, eg, the subject has a congenital immune system deficiency, HIV infection, or is undergoing immunosuppressive therapy.
  • the invention also provides an AIDS immunoassay kit comprising a plurality of components and instructions for indicating an immunization procedure, wherein one component is a live carrier vaccine composition of the invention.
  • Example 1 Construction of recombinant vaccinia virus transfer plasmid expressing HSV-TK gene
  • the herpes simplex virus HSV-1 genomic DNA was used as a template, and the primer HSV-TK-EcoRI/XhoI-up (5'CAA GAATTCCTCGAG TCAGTGGTGGTGGTGGTGGTGGTTAGCCTCCCCC 3' with XbaI, SalI restriction site and 3'-His tag was used.
  • HSV-TK-SalI-for 5'TAC GTCGAC ATGGCTTCGTACCCCTGCCATCAGC 3 ', SEQ ID NO: 11
  • the amplification conditions were as follows: 95 ° C for 5 min; 95 ° C for 30 s, 55 ° C for 30 s, and 72 ° C for 1.5 min for 5 cycles; 95 ° C for 30 s, 60 ° C for 30 s, 72 ° C for 1.5 min, 25 cycles; 72 ° C for 5 min.
  • the HSV-TK fragment is 1149 bp in size.
  • the HSV-TK fragment amplified by PCR was digested with SalI and EcoRI and ligated into the plasmid pSC65 treated with the same restriction enzyme (deposited on February 24, 2004 at the General Microbiology Center of China Microbial Culture Collection Management Committee (CGMCC). The accession number is: CGMCC No. 1097), and the pSC65-TK1 plasmid was obtained.
  • the pSC65-TK1 plasmid was digested with SalI and EcoRI to obtain two fragments of 1351 bp and 4051 bp.
  • the TK gene was sequenced and the results are shown in SEQ ID NO: 3.
  • the plasmid pDRVISV145 2M was used as a template (Escherichia coli containing the plasmid was deposited with CGMCC on May 22, 2008, and the accession number was CGMCC No.2513), with primers gp145 2M for-Sal (5'ACGC GTCGAC AGAGATATCGACACCATGGACAGGG 3 ', SEQ ID NO: 12) and gp145 2M-rev-PacI (5'GCCTTAATTAATCAGTAGCCCTGCCTCACCCTGTTC 3': PCR expansion 13), SEQ ID NO An 2.1 kb HIV-1 gp145 2M gene was added.
  • the PCR reaction conditions were: pre-denaturation at 95 ° C for 5 min; 95 ° C for 30 s, 60 ° C for 30 s, and 72 ° C for 2.5 min for a total of 30 cycles; 72 ° C for 5 min.
  • the amplified HIV-1 gp145 2M fragment and the plasmid pSC65-TK1 were digested with SalI and PacI, and ligated to obtain plasmid pSC65-TK1-gp145 2M, which was identified by double digestion with PacI and XhoI to obtain 4076 bp and 3310 bp. Fragments.
  • the plasmids pSC65-TK1-gp145 2M and pSC65-DR-GN (the plasmid sequence is shown in SEQ ID NO: 4) were digested with PacI and XhoI, and ligated to obtain the transfer plasmid pTK-gp145 2M, which was digested with PacI and XhoI. After getting two 3461 bp and 6231 bp Fragment.
  • the plasmids pSC65-TK1 and pSC65-DR-GN were digested with PacI and XhoI and ligated to obtain the transfer plasmid pTK using the primers HSV-TK-EcoRI/XhoI-up (SEQ ID NO: 10) and HSV-TK- SalI-for (SEQ ID NO: 11) was subjected to PCR amplification and the product was a ⁇ 1.2 kb fragment.
  • Example 2 Construction and identification of recombinant vaccinia virus
  • the TKL and TKR regions of the transfer plasmids pTK and pTK-gp145 2M were homologously recombined with the vaccinia virus Tiantan strain, and the target genes HSV-TK and GP145 2M were recombined with the marker genes neo and GFP into the TK sequence of the vaccinia virus genome.
  • the marker genes neo and GFP are temporarily retained in the genome due to the absence of homologous recombination in the selected pressure molecule, and a recombinant vaccinia virus containing both the gene of interest and green fluorescence can be picked up in a fluorescence microscope (not occurring).
  • the recombinant virus is inhibited from growing due to the presence of G418). Then, under the condition of no G418, the green fluorescent recombinant vaccinia virus itself will lose the neo gene due to the second homologous recombination in the molecule of the TKR-DR fragment of about 200 bp with the complete TKR.
  • a recombinant vaccinia virus containing only the gene of interest is obtained by the GFP gene, and a recombinant virus containing no green fluorescence containing only the gene of interest can be picked.
  • VTT chicken embryo fibroblasts
  • CEF chicken embryo fibroblasts
  • MOI 5% CO 2
  • the recombinant plasmids pTK and pTK-gp145 2M were transfected into cells using liposome, respectively, as described in the kit instructions (Invitrogen, 2000 transfection Reagent, Cat. 11668-027).
  • the recombinant plasmid undergoes homologous recombination with VTT in the cell, and at the same time causes deletion of the VTT thymidine kinase gene. 24 h after transfection, cell-like freeze-thaw was collected 3 times.
  • the pelleted CEF cells were pre-incubated with Eagle's medium containing 0.4 mg/ml G418 for 24 h, and 300 ⁇ l of the transfected virus solution was inoculated into 0.4%/mL G418 pretreated BCR cells for 24 h.
  • the Eagle's medium still contained 0.4 mg/
  • plaques with green fluorescence were picked into 1 ml Eagle's maintenance solution. Repeated freezing and thawing for 3 times, and then 100 ⁇ l was inoculated onto G418-pretreated CEF cells. The above process was repeated, and the single spot was continuously purified for 2 generations to obtain a third generation green fluorescent plaque.
  • the virus picked at this time contains gp145 2M, TK, GFP and neo genes.
  • 100 ⁇ l of CEF cells infected with G418 were inoculated and cultured at 37 ° C for 48 h to pick up non-fluorescent white plaques.
  • 100 ⁇ l of the plaque was inoculated onto G418-pretreated CEF cells. The above procedure was repeated, and the single spot was continuously purified for 5 passages to obtain purified recombinant vaccinia virus TT-TK and TT-EnvTK.
  • TT-TK and TT-EnvTK were deposited with the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee on October 20, 2014.
  • CGMCC General Microbiology Center
  • TT-TK CGMCC No. 9810
  • TT-EnvTK CGMCC No .9808.
  • the genomic DNAs of the recombinant viruses TT-TK and TT-EnvTK were extracted and identified by PCR with the identification primers tkL-up (TAACGTGATGGATATATTAAAGTCG, SEQ ID NO: 14) and tkR-low (AACGACACAACATCCATTTTTAAG, SEQ ID NO: 15).
  • VTT, TT-TK and TT-EnvTK were template fragment sizes of 674 bp, 2830 bp and 4966 bp, respectively, indicating that the gp145 2M and TK genes were correctly recombined into the VTT genome.
  • Recombinant vaccinia virus TT-TK, TT-EnvTK, VTT and VTKgpe were infected with CEF cells at a dose of 1 PFU/cell, respectively. After the hour, the cells were harvested, and 1 ml of protein extraction buffer (1% SDS, 1 mmol/L PMSF, 20 mmol/L Tris-Cl pH 7.0, 1% ⁇ -mercaptoethanol) was added, and immediately mixed, and freeze-thawed three times. PAGE electrophoresis and transfer, 5% skim milk was blocked at room temperature for 2 h.
  • protein extraction buffer 1% SDS, 1 mmol/L PMSF, 20 mmol/L Tris-Cl pH 7.0, 1% ⁇ -mercaptoethanol
  • HSV-TK expression was detected with a murine anti-His-Tag antibody (Gibco, Cat. Fk0168), and HIV-Env expression was detected with HIV-1 SF2 GP160 (from NIH AIDS Research & Reference Reagent Program) antiserum (1:1000 dilution) ).
  • TT-TK and TT-EnvTK had a band of ⁇ 40kD, which was the same size as HSV-TK-His, indicating that the recombinant strain TT-TK can express HSV-TK gene.
  • Both the TT-EnvTK and the positive control detected a ⁇ 140 kD HIV-Env band, indicating that the recombinant strain TT-EnvTK can express the HIV-Env gene.
  • Example 3 Inhibition of recombinant vaccinia virus by ganciclovir (GCV)
  • VTT, TT-TK, and TT-TKEnv were infected with 6-well plate monolayers of CEF or Vero cells with a dose of ⁇ 100 PFU/well, and were injected at 37 ° C, 5% CO 2 for 2 h, and then added with a final concentration of GCV of 0-100 uM.
  • the culture medium was further cultured for 48 hours, and the number of plaques formed in each well was counted, and the inhibition curve of GCV against each recombinant vaccinia virus was plotted. The results are shown in Fig. 1.
  • mice Three-week-old BALB/c mice were randomly divided into 4 ⁇ LD 50 doses of VTT, TT-TK or TT-EnvTK, followed by intraperitoneal injection of GCV at a dose of 80 mg/kg/day/time, and a PBS control group. . Body weight changes and deaths of the mice in each group were recorded daily, and the results are shown in Fig. 2. During the experiment, mice in the PBS control group began to lose weight and activity at 3 days after challenge, and all animals in each group died on the 7th day. The weight loss and death of the mice inoculated with VTT in the GCV group were similar to those in the PBS control group, and all animals died by day 8.
  • VTT and TT-TK were intraperitoneally injected at a dose of 10 7 PFU, and a dose of 80 mg/kg/day was intraperitoneally injected into the GCV and PBS control groups.
  • the bilateral ovaries of each group of mice were taken 8 days after challenge, and the virus titer was titrated on CEF. The results are shown in Figure 3.
  • the degree was as high as 1.2 ⁇ 10 6 PFU and 1 ⁇ 10 6 PFU respectively; the virus titer in the ovary of mice injected with PBS after inoculation with TT-TK was 2 ⁇ 10 3 PFU, but not detected in the ovaries of mice injected with GCV. virus. This result indicates that GCV can inhibit the replication of TT-TK in mouse ovaries.
  • mice Six-week-old female BALB/c mice were randomized into groups of 5 each.
  • the DNA vaccine was intramuscularly injected at 50 ⁇ g/dose/dose at 0, 3, and 6 weeks, and 10 7 PFU TT-EnvTK, and the control strains VTKgpe and VTT, respectively, were boosted at week 9.
  • Mice were sacrificed 1 week after booster immunization, serum was separated, and spleen lymphocytes were prepared.
  • the HIV-specific antibody was detected by ELISA, and the cellular immune response was detected by ELISPOT of IFN- ⁇ .
  • HIV antigen genes in the same recombination region can simplify the virus construction process, but there may be some interference between the foreign genes.
  • gag-specific cellular immune responses induced by the recombinant viruses TT-gag and VTKgpe we compared the gag-specific cellular immune responses induced by the recombinant viruses TT-gag and VTKgpe.
  • the TK region of VTKgpe simultaneously expresses the gag, pol and env genes of HIV, and the recombinant vaccinia virus TT-gag ( deposited on October 20, 2014 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee, deposit number CGMCC No.9809) only expresses the gag gene.
  • CGMCC General Microbiology Center
  • Six-week-old female BALB/c mice were randomized into groups of 5 each.
  • the DNA vaccine was intramuscularly injected at 50 ⁇ g/dose/dose at 0, 3, and 6 weeks, and VTKgpe, TT-gag, and control virus VTT were boosted at week 9 respectively. Mice were sacrificed 1 week after booster immunization, serum was separated, and spleen lymphocytes were prepared.
  • the ELISPOT results of IFN- ⁇ showed that the specific T cell immune response against HIV-1 gag induced by recombinant virus VTKgpe was 330 SFC/10 6 splenocytes, which was significantly lower than TT-gag induced 1038 SFC/10 6 One spleen cell (Figure 5). It can be seen that there is indeed interference between multiple HIV antigen genes in the same recombination region, which reduces the intensity of the immune response. Therefore, the HIV gag and gp145 2M genes should be inserted into different regions of the Tiantan strain, such as the HA region, respectively, to ensure that gag induces stronger cellular immunity.
  • Example 6 Multivalent HIV vaccine expressing different antigens in multiple recombinant regions
  • the genomic DNA of the Tiantan strain vaccinia virus VTT (provided by the Beijing Institute of Biological Products) was used as a template, and the primer A55R-for (5'CATACGCGATCAGAATTCATCGTTGACATCTAGTATTGATAG 3', SEQ with EcoRI restriction enzyme cleavage site was used.
  • ID NO: 16 primer A55R-Rev
  • StuI, XhoI and AscI restriction sites were PCR amplified A55R fragments.
  • the amplification conditions were as follows: 95 ° C for 5 min; 95 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 1 min, 30 cycles; 72 ° C for 5 min.
  • the A55R fragment size is 621 bp.
  • Primer A57R-for (5'GAGAACCTCGAGTTAATTAATGACTTACATAAATGTCTGGGATAG 3', SEQ ID NO: 18) with XhoII and PacI restriction sites and primer A57R-Rev with StuI restriction site (5'TCTAGGCCTTGTTAAAATACATTCTAATACGGTC 3) ', SEQ ID NO: 19) PCR amplification of the A57R fragment.
  • the amplification conditions were as follows: 95 ° C for 5 min; 95 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 1 min, 30 cycles; 72 ° C for 5 min.
  • the A57R fragment is 600 bp in size.
  • Primer A55R-DR-for (5'AAATCTCGAGAGAATTAATCCCGCTCTATGGTCAG 3', SEQ ID NO: 20) with XhoII restriction site and primer A55R-DR-Rev (5' with HindI restriction site) were used.
  • the amplification conditions were as follows: 95 ° C for 5 min; 95 ° C for 30 s, 55 ° C for 30 s, 72 ° C for 30 s, 30 cycles; 72 ° C for 2 min.
  • the A55R-DR fragment is 279 bp in size.
  • the A55R fragment amplified by PCR was digested with XhoI and EcoRI, and the A57R fragment was digested with XhoI and StuI, and ligated into plasmid pUC57 (Genscript, Cat. SD1176) double-digested with XhoI and StuI to obtain pUC57-.
  • A55RA57R plasmid The A55R and A57R fragments were sequenced and the results are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the plasmid pLW73-Neo-I8RDR (the plasmid sequence is shown in SEQ ID NO: 7) was digested with AscI and XhoI to obtain the fragment Neo-GFP.
  • the fragment Neo-GFP was ligated into the plasmid pUC57-A55RA57R digested with the same restriction endonuclease to obtain the transfer plasmid pUC57-A55RA57R-GN, and the plasmid size was 5763 bp.
  • the PCR amplified fragment A55R-DR was digested with HindIII and XhoI, and cloned into the plasmid pSC65 digested with the same restriction endonuclease to obtain a recombinant plasmid pSC65-A55R-DR.
  • the A55R-DR fragment was sequenced and the results are shown in SEQ ID NO:8.
  • Plasmid pSC65-A55R-DR was digested with PacI and XhoI to obtain fragment pE/L-DR, ligated into plasmid pUC57-A55RA57R-GN digested with PacI and XhoI to obtain plasmid pVI76, and the plasmid size was 6117 bp.
  • the plasmid was sequenced and the results are shown in SEQ ID NO:9.
  • Gag fragment (SEQ ID NO: 2) and plasmid pVI76 were digested with KpnI and PacI Then, the transfer plasmid pVI76-gag was obtained. Double digestion with KpnI and PacI endonuclease showed that the size of the digested products were 1488 bp and 6117 bp, respectively, indicating that the Gag gene was correctly inserted into plasmid pVI76.
  • the recombinant plasmid pVI76-gag was transfected into the cells as described in the kit instructions (Invitrogen, 2000 transfection Reagent, Cat. 11668-027).
  • the recombinant plasmid is homologously recombined in the HA region with TT-EnvTK in the cell. 24 h after transfection, cell-like freeze-thaw was collected 3 times.
  • the recombinant virus screening method was the same as that in Example 2, and purified by 3 generations of green fluorescent plaques and 5 generations of white plaques to obtain purified recombinant vaccinia virus TT-TK+/EG.
  • the genomic DNA of the recombinant virus TT-TK+/EG was extracted using the identification primers tkL-up (SEQ ID NO: 14) and tkR-low (SEQ ID NO: 15), SQEA57R-rev (5'TGTTAAAATACATTCTAATACGGTC 3', SEQ ID NO :22) and SQE55R-for (5'ATCGTTGACATCTAGTATTGATAG 3', SEQ ID NO: 23) were identified by PCR.
  • the results showed that the amplified fragments of the two pairs of primers were 4966 bp and 3047 bp, respectively, indicating that gp145 2M and HSV-TK were correctly recombined.
  • the Gag gene was correctly recombined into the VTT genomic HA region.
  • clones of recombinant vaccinia virus TT-TK+/EG were infected with CEF cells at a dose of 1 PFU/cell, and cells were harvested 24 hours later, and 1 ml of protein extraction buffer (1% SDS, 1 mmol/L PMSF, 20 mmol) was added. /L Tris-Cl pH 7.0, 1% ⁇ -mercaptoethanol), mix immediately, and freeze-thaw three times. PAGE electrophoresis and transfer, 5% skim milk was blocked at room temperature for 2 h.
  • HIV-1 Gp145 2M and Gag The expression of HIV-1 Gp145 2M and Gag was detected with HIV-1 SF2 GP160 antiserum and HIV-1 Gag antiserum (both from NIH AIDS Research & Reference Reagent Program).
  • HIV-1 SF2 GP160 antiserum and HIV-1 Gag antiserum both from NIH AIDS Research & Reference Reagent Program.
  • the membrane was washed 3 times with PBS for 10 min each time.
  • the horseradish peroxidase-labeled secondary antibody, 5% skim milk was diluted 1:5000 and incubated for 1 h at room temperature.
  • the membrane was washed 3 times with PBS for 10 min each time.
  • Kern E R In vitro activity of potential anti-poxvirus agents. Antiviral Res, 2003. 57 (1-2): 35-40.

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Abstract

提供一种重组复制型痘苗病毒，所述病毒包含编码HSV-TK和HIV抗原的多核苷酸。还提供了一种活载体HIV疫苗组合物，其包含所述重组复制型痘苗病毒。

Description

复制型痘苗病毒载体艾滋病疫苗 技术领域
本发明涉及医学生物技术领域，具体涉及复制型痘苗病毒艾滋病疫苗，其能够诱导高水平的抗人类免疫缺陷病毒的体液和细胞免疫应答，并允许对疫苗可能导致的严重不良反应进行有效控制。
背景技术
痘苗病毒载体是研究最为深入和广泛的病毒载体之一，可作为用于构建疫苗的载体。非复制型痘苗病毒载体的安全性高，但免疫原性相对较弱，特别是在人体中诱导的免疫反应明显弱于猴体内的免疫应答。复制型痘苗病毒载体免疫原性强，可诱导持久的免疫力，但具有引起严重的不良反应等潜在安全性问题。
由于天花已经消灭了多年，作为天花疫苗的痘苗病毒已经停止接种30多年，针对痘苗病毒的药物研发已很少有人关注。随着复制型痘苗病毒载体在疫苗开发领域中的应用，亟需在出现严重不良反应时可以进行有效治疗的药物和措施。
发明内容
在第一方面，本发明提供一种重组复制型痘苗病毒，其在TK区包含编码HSV-TK的第一多核苷酸和编码HIV抗原的第二多核苷酸。
优选地，本发明的重组复制型痘苗病毒是痘苗病毒天坛株。
在一个具体的实施方式中，本发明的重组复制型痘苗病毒中的所述第一多核苷酸包含SEQ ID NO:3所示的核苷酸序列。在另一实施方式中，本发明的重组复制型痘苗病毒中的所述第一多核苷酸包含与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第一多核苷酸编码来自HSV-1的TK。
在另一个具体的实施方式中，本发明的重组复制型痘苗病毒中的所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp145 2M并包含SEQ ID NO:1所示的核苷酸序列。在另一实施方式中，本发 明的重组复制型痘苗病毒中的所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp145 2M或其免疫原性片段。在另一实施方式中，本发明的重组复制型痘苗病毒中的所述第二多核苷酸包含与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第二多核苷酸编码来自HIV-1的gp145 2M。
优选地，本发明的重组复制型痘苗病毒还包含编码额外的HIV抗原的第三多核苷酸。在一个优选的实施方式中，所述第三多核苷酸插入复制型痘苗病毒的至HA区。在一个具体的实施方式中，所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag并包含SEQ ID NO:2所示的核苷酸序列。在另一实施方式中，所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag或其免疫原性片段。在另一实施方式中，所述第三多核苷酸包含与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第三多核苷酸编码来自HIV-1的gag。
在一个优选的实施方式中，本发明提供重组复制型痘苗病毒，其是天坛株痘苗病毒，其包含插入至天坛株痘苗病毒的TK区的编码来自中国HIV主要流行毒株97CN001的gp145 2M的基因和编码HSV-TK的基因，并包含插入至天坛株痘苗病毒的HA区的编码97CN001的gag的基因。
本发明进一步提供活载体HIV疫苗组合物，其包含上述第一方面的本发明的重组复制型痘苗病毒和药用可接受载体和/或佐剂。
本发明还涉及预防和/或治疗对象的HIV感染的方法，包括给所述对象施用包含上述第一方面的本发明的重组复制型痘苗病毒的活载体HIV疫苗组合物。本发明还涉及上述第一方面的本发明的重组复制型痘苗病毒在制备用于预防和/或治疗对象的HIV感染的活载体HIV疫苗组合物中的用途。优选地，所述对象是免疫抑制患者，例如所述对象患有先天性免疫系统缺陷、HIV感染或正接受免疫抑制治疗。
本发明还提供一种艾滋病免疫试剂盒，所述试剂盒包含多个成份和指示免疫程序的说明书，其中一个成份为本发明的活载体疫苗组合物。
附图说明
图1：GCV对重组痘苗病毒的体外抑制作用。
图2：GCV对小鼠脑内TT-TK和TT-EnvTK复制的抑制作用。
图3：GCV对小鼠卵巢中TT-TK复制的抑制作用。
图4：TT-EnvTK在小鼠诱导的免疫反应。A：细胞免疫；B：体液免疫。
图5：多个HIV抗原间的免疫干扰。
保藏信息
重组痘苗病毒TT-TK于2014年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.9810；
重组痘苗病毒TT-EnvTK于2014年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.9808；
重组痘苗病毒TT-gag于2014年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.9809。
序列信息
SEQ ID NO:1：Gp145 2M基因序列，来自HIV-1；
SEQ ID NO:2：Gag基因序列，来自HIV-1；
SEQ ID NO:3：HSV-TK基因序列，来自HSV-1；
SEQ ID NO:4：质粒pSC65-DR-GN序列。
具体实施方案
获得性免疫缺陷综合征(Acquired Immunodeficiency Syndrome，AIDS)简称艾滋病，是由人免疫缺陷病毒(Human Immunodeficiency Virus，HIV)感染引起的一种传染性疾病。自从1981年发现第一例病例以来，艾滋病一直以惊人的速度在全球蔓延，成为世界上危害人类生命和健康最严重的病毒性疾病之一。艾滋病的流行给世界的社会和经济发展带来了巨大影响。在一些发展中国家，HIV感染已造成了人均寿命缩短、劳动力减少、食品短缺，使经济和社会发展倒退了20年。HIV在 中国的流行经历了传入期(1985－1988)、播散期(1989－1994)和增长期(1995年至今)三个阶段。
近年来抗HIV病毒药物的研制和应用取得了很大进展，部分感染者得到了较好的治疗。然而，耐药株的出现使这些药物不是对每个病人都有效，复杂的服药过程使部分病人不能坚持按时服药而导致治疗失败，昂贵的费用也使治疗难以大规模推广。历史经验证明，控制疾病流行最经济有效的方法就是应用疫苗，成功的实例如天花、脊髓灰质炎、麻疹、肝炎等，因此，研制安全有效的艾滋病疫苗一直是科学工作者们奋斗的目标。
各国科学家一致认为有效的艾滋病疫苗必须能够诱导机体产生HIV特异性的CD8+细胞毒T淋巴细胞反应(CTL)和中和抗体反应。目前各单一疫苗难以实现这一目标，需要采用不同类型疫苗联合免疫的策略才能提高艾滋病疫苗的免疫效果。可供选择的候选疫苗包括下列几种：传统疫苗(灭活疫苗和减毒活疫苗)、合成肽和蛋白亚单位疫苗、DNA疫苗以及活载体疫苗。
与其它类型疫苗相比，活载体疫苗的优势体现在：(1)能主动感染靶组织或细胞，提高了外源基因进入细胞的效率；(2)载体自身有佐剂效应，能诱导细胞因子和趋化因子的产生；(3)多数能诱导长期的免疫应答。
其中以痘苗病毒为载体的活载体疫苗研究进行得最为深入和广泛，但目前进入临床试验阶段的多为非复制型载体，如MVA、NYVAC和ALVAC。但临床试验显示非复制型载体免疫原性相对较弱，而且同一种疫苗在人体中诱导的免疫反应明显弱于猴体内的免疫应答。这可能与其非复制的特性有关。非复制型载体疫苗感染宿主细胞后，只能进行一个周期的抗原表达、加工和呈递过程，无法产生有感染性的病毒并开始新一轮的感染，因而它对免疫系统的刺激是有限的和短暂的。有鉴于此，国际上活载体疫苗的研究重点已从非复制型转向复制型，以充分发挥活载体能够复制的特点，更好地刺激机体产生免疫反应。
复制型痘苗病毒载体免疫原性强，可诱导持久的免疫力，但具有引起严重的不良反应等潜在安全性问题。特别是对于因先天性免疫系统缺陷、免疫抑制治疗或HIV感染等原因造成免疫系统抑制的个体，复制型痘苗病毒载体疫苗可能会引起严重的不良反应[1,2]。
由于天花已经消灭了多年，作为天花疫苗的痘苗病毒已经停止接种30多年，针对痘苗病毒的药物研发已很少有人关注。随着复制型痘苗病毒载体在疫苗开发领域中的应用，亟需在出现严重不良反应时可以进行有效治疗的药物和措施。
自杀基因系统是近些年来广泛用于癌症等疾病治疗的方法，常见的自杀基因/药物组合有：胞嘧啶脱氨酶(Cytosine deaminase,CD)/5-氟胞嘧啶(5-fluorocytosine)[3]、硝基还原酶(Nitroreductase)/CB 1954[4]、胸苷磷酸化酶(Thymidine phosphorylase,TP)/5'脱氧5氟尿嘧啶(5'-deoxy-5-fluorouridine,5'-DFUR)[5]、嘌呤核苷磷酸化酶(Purine nucleoside phosphorylase,PNP)/6甲基嘌呤2'脱氧核苷(6-methylpurine-2'-deoxyriboside,MeP-dR)[6]和单纯疱疹病毒胸苷激酶(Herpes simplex virus thymidine kinase,HSV-TK)/更昔洛韦(Ganciclovir,GCV)[7]。其中HSV-TK/GCV是研究并应用最多的自杀基因系统。
GCV活性的发挥特异性依赖于HSV的TK基因，HSV TK可以磷酸化GCV，生成其单磷酸形式(GCV-MP)[8]，接着细胞激酶进一步将GCV-MP磷酸化，生成二磷酸盐(GCV-DP)和有毒性的三磷酸形式(GCV-TP)。GCV-TP是一个DNA聚合酶抑制剂，可以通过竞争抑制脱氧鸟苷三磷酸与DNA聚合酶的结合或插入到新生链中的方式抑制DNA复制，造成新链延长的终止，最后使细胞死亡[9]。由于GCV无法直接被哺乳动物细胞激酶磷酸化，药物前体只对表达HSV-TK的细胞存在选择性毒性，不良反应很小。
目前已有报道使用携带HSV-TK/GCV自杀基因系统的病毒载体来治疗肿瘤，其中HSV-TK/GCV自杀基因系统的作用是直接杀伤靶细胞，而非用于保证活病毒载体使用过程中的安全性并控制可能发生的疫苗载体相关性不良反应。此外，目前HSV-TK/GCV自杀基因系统主要用于腺病毒载体和腺相关病毒载体，HSV-TK/GCV系统与痘苗病毒载体，特别是作为复制型痘苗病毒载体的痘苗病毒天坛株之间的相容性尚未证实。痘苗病毒的TK基因与HSV-TK同源性只有～16％，更昔洛韦对痘苗病毒似乎没有抑制作用(EC50>300μM)[10]。
本发明人将HSV-TK基因引入复制型痘苗病毒载体中，从而提供了一种安全可控的复制型痘苗病毒载体艾滋病疫苗。本发明人证实，将HSV-TK基因引入到复制型痘苗病毒艾滋病疫苗，在不影响复制型痘病 毒载体免疫原性的前提下，有效控制了复制型痘苗病毒载体可能导致的严重不良反应，提高了疫苗的安全性，解决了复制型痘苗病毒载体的安全性问题。本发明的复制型痘苗病毒载体艾滋病疫苗特别适用于免疫抑制患者，例如患有先天性免疫系统缺陷、HIV感染或正接受免疫抑制治疗的对象。
对艾滋病疫苗而言，HIV抗原的选择也非常重要。HIV特异性的CD8+T细胞杀伤作用(CTL)在控制HIV复制中发挥了重要作用。Gag特异性CTL对感染者控制病毒复制是有利的，Env诱导的中和抗体可以中和HIV，V1V2结合抗体滴度与疫苗的免疫保护正相关。因此艾滋病疫苗应该选择多个抗原，以诱导不同类型的免疫应答。不仅如此，本发明人发现，将HIV-1 gag和gp145 2M基因分别引入复制型痘苗病毒载体的HA区和TK区，在保证疫苗表达多个抗原的前提下，还避免了不同抗原之间的干扰。
因此，在第一方面，本发明提供一种重组复制型痘苗病毒，其在TK区包含编码HSV-TK的第一多核苷酸和编码HIV抗原的第二多核苷酸。
优选地，本发明的重组复制型痘苗病毒是痘苗病毒天坛株。
在一个具体的实施方式中，本发明的重组复制型痘苗病毒中的所述第一多核苷酸包含SEQ ID NO:3所示的核苷酸序列。在另一实施方式中，本发明的重组复制型痘苗病毒中的所述第一多核苷酸包含与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第一多核苷酸编码来自HSV-1的TK。
在另一个具体的实施方式中，本发明的重组复制型痘苗病毒中的所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp145 2M并包含SEQ ID NO:1所示的核苷酸序列。在另一实施方式中，本发明的重组复制型痘苗病毒中的所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp145 2M或其免疫原性片段。在另一实施方式中，本发明的重组复制型痘苗病毒中的所述第二多核苷酸包含与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第二多核苷酸编码来自 HIV-1的gp145 2M。
优选地，本发明的重组复制型痘苗病毒还包含编码额外的HIV抗原的第三多核苷酸。在一个优选的实施方式中，所述第三多核苷酸插入复制型痘苗病毒的至HA区。在一个具体的实施方式中，所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag并包含SEQ ID NO:2所示的核苷酸序列。在另一实施方式中，所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag或其免疫原性片段。在另一实施方式中，所述第三多核苷酸包含与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第三多核苷酸编码来自HIV-1的gag。
在一个优选的实施方式中，本发明提供重组复制型痘苗病毒，其是天坛株痘苗病毒，其包含插入至天坛株痘苗病毒的TK区的编码来自中国HIV主要流行毒株97CN001的gp145 2M的基因和编码HSV-TK的基因，并包含插入至天坛株痘苗病毒的HA区的编码97CN001的gag的基因。
本发明进一步提供活载体HIV疫苗组合物，其包含上述第一方面的本发明的重组复制型痘苗病毒和药用可接受载体和/或佐剂。
本发明还涉及预防和/或治疗对象的HIV感染的方法，包括给所述对象施用包含上述第一方面的本发明的重组复制型痘苗病毒的活载体HIV疫苗组合物。本发明还涉及上述第一方面的本发明的重组复制型痘苗病毒在制备用于预防和/或治疗对象的HIV感染的活载体HIV疫苗组合物中的用途。优选地，所述对象是免疫抑制患者，例如所述对象患有先天性免疫系统缺陷、HIV感染或正接受免疫抑制治疗。
本发明还提供一种艾滋病免疫试剂盒，所述试剂盒包含多个成份和指示免疫程序的说明书，其中一个成份为本发明的活载体疫苗组合物。
实施例
实施例1：表达HSV-TK基因重组痘苗病毒转移质粒的构建
转移质粒pVI75-GFP-TK和pVI75-GFP-EnvTK的构建方法具体如下：
1.质粒pSC65-TK1的构建
以单纯疱疹病毒HSV-1基因组DNA为模板，用带有XbaI、SalI限制性酶切位点以及3’端His标签的引物HSV-TK-EcoRI/XhoI-up(5'CAAGAATTCCTCGAGTCAGTGGTGGTGGTGGTGGTGGTTAGCCTCCCCC 3'，SEQ ID NO:10)和HSV-TK-SalI-for(5’TACGTCGACATGGCTTCGTACCCCTGCCATCAGC 3’，SEQ ID NO:11)PCR扩增HSV-TK基因。扩增条件如下：95℃ 5min；95℃ 30s，55℃ 30s，72℃ 1.5min，进行5个循环；95℃ 30s，60℃ 30s，72℃ 1.5min，25个循环；72℃ 5min。HSV-TK片段大小为1149bp。
将PCR扩增得到的HSV-TK片段经过SalI和EcoRI双酶切后连入用相同酶切处理的质粒pSC65(2004年2月24日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号是：CGMCC No.1097)，得到pSC65-TK1质粒。pSC65-TK1质粒经SalI和EcoRI双酶切鉴定，得到1351bp和4051bp两个片段。对TK基因进行测序，结果示于SEQ ID NO:3。
2.质粒pSC65-TK1-gp145 2M的构建
为了构建同时表达HSV-TK和HIV-1 gp145 2M的重组质粒pSC65-TK1-gp145 2M，以质粒pDRVISV145 2M为模板(含有该质粒的大肠杆菌于2008年5月22日保藏于CGMCC，保藏号为CGMCC No.2513)，用引物gp145 2M for-Sal(5'ACGCGTCGACAGAGATATCGACACCATGGACAGGG 3'，SEQ ID NO:12)和gp145 2M–rev-PacI(5'GCCTTAATTAATCAGTAGCCCTGCCTCACCCTGTTC 3'，SEQ ID NO:13)PCR扩增2.1kb的HIV-1 gp145 2M基因。PCR反应条件为：95℃预变性5min；95℃ 30s，60℃ 30s，72℃ 2.5min，共进行30个循环；72℃充分延伸5min。将扩增得到的HIV-1 gp145 2M片段和质粒pSC65-TK1经过SalI和PacI双酶切后进行连接，得到质粒pSC65-TK1-gp145 2M，其PacI和XhoI双酶切鉴定，获得4076bp和3310bp两个片段。
3.转移质粒pTK-gp145 2M和pTK的构建
质粒pSC65-TK1-gp145 2M和pSC65-DR-GN(质粒序列见SEQ ID NO:4)用PacI和XhoI进行双酶切后进行连接，得到转移质粒pTK-gp145 2M，其经PacI和XhoI酶切后得到3461bp和6231bp两个 片段。
质粒pSC65-TK1和pSC65-DR-GN用PacI和XhoI进行双酶切后进行连接，得到转移质粒pTK，采用引物HSV-TK-EcoRI/XhoI-up(SEQ ID NO:10)和HSV-TK-SalI-for(SEQ ID NO:11)进行PCR扩增，产物为～1.2kb片段。
实施例2：重组痘苗病毒的构建和鉴定
转移质粒pTK和pTK-gp145 2M的TKL和TKR区与痘苗病毒天坛株发生同源重组，使目的基因HSV-TK和GP145 2M与标记基因neo和GFP一同重组到痘苗病毒基因组的TK序列中。在G418存在的条件下，由于选择压力分子内未发生同源重组，标记基因neo和GFP暂时保留在基因组中，在荧光显微镜可以挑取既含目的基因又带绿色荧光的重组痘苗病毒(未发生重组的病毒因G418的存在而被抑制生长)。接着在无G418的条件下筛选，带绿色荧光重组痘苗病毒自身会因为转移质粒带入的一小段约200bp的TKR-DR片段与完整的TKR发生分子内的二次同源重组，从而丢失neo基因和GFP基因而得到了只含目的基因的重组痘苗病毒,就能挑取只含目的基因的无绿色荧光的重组病毒。
1.重组痘苗病毒的构建和纯化
天坛株痘苗病毒VTT(该病毒由北京生物制品研究所提供)以MOI＝1的接毒量感染80-90％成片的鸡胚成纤维细胞(CEF)，37℃，5％CO2培养2h，利用脂质体将重组质粒pTK和pTK-gp145 2M分别转染进入细胞，方法见试剂盒说明书(Invitrogen,
Figure PCTCN2015092170-appb-000001
2000 transfection Reagent,Cat.11668-027)。重组质粒在细胞内与VTT发生同源重组，同时造成VTT胸苷激酶基因的缺失。转染后24h，收取细胞样冻融3次。
成片的CEF细胞先用含0.4mg/ml G418的Eagle’s培养基预培养24h，收获的转染病毒液300μl接种到0.4mg/mL G418预处理24h的CEF细胞，Eagle’s培养基仍含有0.4mg/mL G418和1％低熔点琼脂糖，37℃培养48h后，挑取带有绿色荧光的噬斑至1ml Eagle’s维持液中。反复冻融3次，再取100μl接种到G418预处理的CEF细胞上。重复上述过程，单斑连续纯化2代，得到第三代绿色荧光噬斑。此时挑取的病毒含有gp145 2M、TK、 GFP和neo基因。将第三代绿色荧光噬斑冻融三次后，分别取100μl感染未经G418处理的CEF细胞，37℃培养48h，挑取无荧光的白色噬斑。白色噬斑病毒冻融3次后，再取100μl接种到G418预处理的CEF细胞上。重复上述过程，单斑连续纯化5代，得到纯化的重组痘苗病毒TT-TK和TT-EnvTK。TT-TK和TT-EnvTK于2014年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号分别是：TT-TK：CGMCC No.9810，TT-EnvTK：CGMCC No.9808。
2.重组病毒的PCR鉴定
提取重组病毒TT-TK和TT-EnvTK的基因组DNA，用鉴定引物tkL-up(TAACGTGATGGATATATTAAAGTCG，SEQ ID NO:14)和tkR-low(AACGACACAACATCCATTTTTAAG，SEQ ID NO:15)进行PCR鉴定，结果显示，以VTT、TT-TK和TT-EnvTK为模板片段大小分别为674bp；，2830bp和4966bp，表明gp145 2M和TK基因均正确地重组到VTT基因组上。
3.重组痘苗病毒目的基因的表达
重组痘苗病毒TT-TK、TT-EnvTK、VTT和VTKgpe(已在中国微生物菌种保藏管理委员会普通微生物中心进行保藏，编号为CGMCC NO.1099)分别以1PFU/cell的剂量感染CEF细胞,培养24小时后收获细胞，加入1ml的蛋白提取缓冲液(1％SDS，1mmol/L PMSF,20mmol/L Tris-Cl pH7.0,1％β-巯基乙醇)，立即混匀，反复冻融三次。PAGE电泳并转膜，5％的脱脂奶室温封闭2h。用鼠抗His-Tag抗体(Gibco，Cat.Fk0168)检测HSV-TK的表达，用HIV-1 SF2 GP160(来自NIH AIDS Research & Reference Reagent Program)抗血清检测HIV-Env的表达(1:1000稀释)。5％的脱脂奶稀释一抗室温孵育2h。PBS洗膜3次，每次10min。加入相应的辣根过氧化物酶标记的二抗，5％的脱脂奶按1:5000稀释，室温孵育1h。PBS洗膜3次，每次10min。ECL显色后,结果显示，TT-TK和TT-EnvTK有一条～40kD的条带，其大小与HSV-TK-His相同，这说明了重组毒株TT-TK可以表达HSV-TK基因。TT-EnvTK和阳性对照都可以检测到～140kD的HIV-Env条带，这说明重组毒株TT-EnvTK可以表达HIV-Env基因。
实施例3：更昔洛韦(GCV)对重组痘苗病毒的抑制作用
1.GCV对重组痘苗病毒的体外抑制实验
VTT、TT-TK、TT-TKEnv分别以～100PFU/孔的接毒量感染6孔板单层CEF或Vero细胞，37℃，5％CO2感染2h后加入含有GCV终浓度为0～100uM的培养基，继续培养48h后统计各孔中形成的噬斑数量，绘制GCV对各重组痘苗病毒的抑制曲线，结果如图1所示。在CEF(A)和Vero(B)中，GCV对TT-TK和TT-EnvTK有明显抑制作用，TT-TK和TT-EnvTK的病毒噬斑数均与GCV浓度呈负相关。GCV对VTT则没有抑制作用。
2.GCV对重组痘苗病毒的体内抑制实验
3周龄BALB/c小鼠随机分组，脑内接种10×LD50剂量的VTT、TT-TK或TT-EnvTK，随后以80mg/kg/天/次的剂量腹腔注射GCV，同时设PBS对照组。每天记录各组中小鼠的体重变化和死亡情况，结果如图2所示。实验过程中，PBS对照组的小鼠在攻毒后3天开始出现体重和活动能力的下降，到第7天各组中的动物全部死亡。接种VTT的GCV注射组小鼠的体重下降和死亡情况与PBS对照组相似，至第8天所有动物均死亡。接种TT-TK和TT-EnvTK的GCV注射组中的动物的体重略有下降，第3天开始逐渐恢复，且动物全部存活。这一结果证明，GCV可以抑制TT-TK和TT-EnvTK在小鼠脑内的复制。
8周龄雌性C57BL小鼠随机分组，VTT和TT-TK以107PFU的剂量进行腹腔注射，分别设80mg/kg/天的剂量腹腔注射GCV和PBS对照组。于攻毒后8天取各组小鼠的双侧卵巢，在CEF上对其中的病毒滴度进行滴定，结果如图3所示，接种VTT后注射PBS或GCV的小鼠卵巢中的病毒滴度分别高达1.2×106PFU和1×106PFU；接种TT-TK后注射PBS的小鼠卵巢中的病毒滴度为2×103PFU，而注射GCV的小鼠卵巢中则检测不到病毒。此结果说明GCV可以抑制TT-TK在小鼠卵巢中的复制。
实施例4：重组痘苗病毒免疫原性研究
6周龄雌性BALB/c小鼠随机分组，每组5只。在0，3，6周肌肉注射DNA疫苗50μg/只/剂，第9周分别加强免疫107PFU TT-EnvTK，以及对照毒株VTKgpe和VTT。加强免疫后1周处死小鼠，分离血清、制备脾淋巴 细胞。ELISA方法检测HIV特异性抗体，IFN-γ的ELISPOT检测细胞免疫反应。结果显示，重组病毒TT-EnvTK诱导产生的针对HIV-Env的特异性T细胞免疫反应强度为956 SFC/106个脾细胞，而对照疫苗株VTKgpe为996SFC/106个脾细胞；统计学分析显示，两组之间没有显著性差异(P>0.05)(图4A)。TT-EnvTK诱导出的抗体滴度为104.7，VTKgpe的为104.5。统计学分析表明，两组之间无显著性差异(P>0.05)(图4B)。实验结果表明，TT-EnvTK能够诱导HIV特异性细胞和体液免疫反应，且免疫反应强度与对照病毒无显著差异。
实施例5：同一重组区内多个HIV抗原基因的相互干扰
在同一重组区表达多个HIV抗原基因可以简化病毒构建过程，但外源基因之间可能存在一定的干扰现象。为此，我们比较了重组病毒TT-gag和VTKgpe诱导的gag特异性细胞免疫反应。
VTKgpe的TK区同时表达了HIV的gag,pol和env基因，而重组痘苗病毒TT-gag(于2014年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏号CGMCC No.9809)仅表达gag基因。6周龄雌性BALB/c小鼠随机分组，每组5只。在0，3，6周肌肉注射DNA疫苗50μg/只/剂，第9周分别加强免疫VTKgpe、TT-gag和对照病毒VTT。加强免疫后1周处死小鼠，分离血清、制备脾淋巴细胞。IFN-γ的ELISPOT结果显示，重组病毒VTKgpe诱导产生的针对HIV-1 gag的特异性T细胞免疫反应强度为330 SFC/106个脾细胞，显著低于TT-gag诱导的1038 SFC/106个脾细胞(图5)。由此可以看出，同一重组区内多个HIV抗原基因之间确实存在干扰现象，降低免疫应答的强度。因此，HIVgag、gp145 2M基因应该分别插入天坛株的不同区域，如HA区，以确保gag诱导更强的细胞免疫。
实施例6：在多个重组区表达不同抗原的多价HIV疫苗
1.转移质粒pVI76-gag的构建
以天坛株痘苗病毒VTT(该病毒由北京生物制品研究所提供)基因组DNA为模板，用带有EcoRI限制性酶切位点的引物A55R-for(5'CATACGCGATCAGAATTCATCGTTGACATCTAGTATTGATAG 3'，SEQ  ID NO:16)和带有StuI、XhoI和AscI限制性酶切位点的引物A55R-Rev(5’TAAGGCCTCTCGAGGCGCGCCCTATCAACTACCTATAAAACTTTCC3’，SEQ ID NO:17)PCR扩增A55R片段。扩增条件如下：95℃ 5min；95℃ 30s，55℃ 30s，72℃ 1min，30个循环；72℃ 5min。A55R片段大小为621bp。用带有XhoII和PacI限制性酶切位点的引物A57R-for(5'GAGAACCTCGAGTTAATTAATGACTTACATAAATGTCTGGGATAG 3'，SEQ ID NO:18)和带有StuI限制性酶切位点的引物A57R-Rev(5’TCTAGGCCTTGTTAAAATACATTCTAATACGGTC 3’，SEQ ID NO:19)PCR扩增A57R片段。扩增条件如下：95℃ 5min；95℃ 30s，55℃ 30s，72℃ 1min，30个循环；72℃ 5min。A57R片段大小为600bp。用带有XhoII限制性酶切位点的引物A55R-DR-for(5'AAATCTCGAGAGAATTAATCCCGCTCTATGGTCAG 3'，SEQ ID NO:20)和带有HindI限制性酶切位点的引物A55R-DR-Rev(5’GCGAAGCTTTTGTTCTATCAACTACCTATAAAAC 3’，SEQ ID NO:21)PCR扩增A55R-DR片段。扩增条件如下：95℃ 5min；95℃ 30s，55℃ 30s，72℃ 30s，30个循环；72℃ 2min。A55R-DR片段大小为279bp。
将PCR扩增得到的A55R片段经过XhoI和EcoRI双酶切、A57R片段经过XhoI和StuI双酶切后连入用XhoI和StuI双酶切处理的质粒pUC57(Genscript，Cat.SD1176)，得到pUC57-A55RA57R质粒。对A55R和A57R片段进行测序，结果分别示于SEQ ID NO:5和SEQ ID NO:6。
质粒pLW73-Neo-I8RDR(质粒序列见SEQ ID NO:7)用AscI和XhoI双酶切后获得片段Neo-GFP。片段Neo-GFP连接到经相同限制性内切酶消化的质粒pUC57-A55RA57R中，得到转移质粒pUC57-A55RA57R-GN，质粒大小为5763bp。
PCR扩增片段A55R-DR经HindIII和XhoI双酶切后，克隆入经相同限制性内切酶消化的质粒pSC65中，获得重组质粒pSC65-A55R-DR。对A55R-DR片段进行测序，结果示于SEQ ID NO:8。质粒pSC65-A55R-DR用PacI和XhoI双酶切后获得片段pE/L-DR，连接入经PacI和XhoI双酶切的质粒pUC57-A55RA57R-GN，获得质粒pVI76，质粒大小为6117bp。对质粒进行测序，结果示于SEQ ID NO:9。
Gag片段(SEQ ID NO:2)和质粒pVI76经KpnI和PacI双酶切后连 接，获得转移质粒pVI76-gag。用KpnI和PacI内切酶进行双酶切验证，结果表明，酶切产物大小分别为1488bp和6117bp，表明Gag基因被正确插入质粒pVI76中。
2.重组病毒的构建和纯化
重组病毒TT-EnvTK(CGMCC No.9808)以MOI＝1的接毒量感染80-90％成片的鸡胚成纤维细胞(CEF)，37℃，5％CO2培养2h，利用脂质体将重组质粒pVI76-gag转染进入细胞，方法见试剂盒说明书(Invitrogen,
Figure PCTCN2015092170-appb-000002
2000 transfection Reagent,Cat.11668-027)。重组质粒在细胞内与TT-EnvTK在HA区发生同源重组。转染后24h，收取细胞样冻融3次。
重组病毒筛选方法同实施例2，经3代绿色荧光噬斑、5代白色噬斑筛选纯化，得到纯化的重组痘苗病毒TT-TK+/EG。
3.重组病毒的PCR鉴定
提取重组病毒TT-TK+/EG的基因组DNA，用鉴定引物tkL-up(SEQ ID NO:14)和tkR-low(SEQ ID NO:15)，SQEA57R-rev(5’TGTTAAAATACATTCTAATACGGTC 3’，SEQ ID NO:22)和SQE55R-for(5’ATCGTTGACATCTAGTATTGATAG 3’，SEQ ID NO:23)进行PCR鉴定，结果显示，两对引物扩增片段大小分别为4966bp和3047bp，表明gp145 2M、HSV-TK正确地重组到VTT基因组TK区，Gag基因正确地重组到VTT基因组HA区。
4.重组痘苗病毒目的基因的表达
重组痘苗病毒TT-TK+/EG的1-5#克隆分别以1PFU/cell的剂量感染CEF细胞,培养24小时后收获细胞，加入1ml的蛋白提取缓冲液(1％SDS，1mmol/L PMSF,20mmol/L Tris-Cl pH7.0,1％β-巯基乙醇)，立即混匀，反复冻融三次。PAGE电泳并转膜，5％的脱脂奶室温封闭2h。用HIV-1 SF2 GP160抗血清和HIV-1 Gag抗血清(均来自NIH AIDS Research & Reference Reagent Program)分别检测HIV-1 Gp145 2M和Gag的表达。5％的脱脂奶稀释一抗室温孵育2h。PBS洗膜3次，每次10min。加入相应 的辣根过氧化物酶标记的二抗，5％的脱脂奶按1:5000稀释，室温孵育1h。PBS洗膜3次，每次10min。ECL显色后,结果显示，1-5#重组病毒均可检测到～140kD的HIV-1 Gp145 2M条带和～55kD的HIV-1 Gag条带，这说明重组毒株可以正确表达Gp145 2M和Gag基因。
参考文献
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Claims (18)

  1. 重组复制型痘苗病毒，其在TK区包含编码HSV-TK的第一多核苷酸和编码HIV抗原的第二多核苷酸。
  2. 权利要求1的重组复制型痘苗病毒，其中所述复制型痘苗病毒是痘苗病毒天坛株。
  3. 权利要求1或2的重组复制型痘苗病毒，其中所述第一多核苷酸包含SEQ ID NO:3所示的核苷酸序列。
  4. 权利要求1或2的重组复制型痘苗病毒，其中所述第一多核苷酸包含与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第一多核苷酸编码来自HSV-1的TK。
  5. 权利要求1-4任一项的重组复制型痘苗病毒，其中所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp1452M并包含SEQ ID NO:1所示的核苷酸序列。
  6. 权利要求1-4任一项的重组复制型痘苗病毒，其中所述第二多核苷酸编码来源于中国HIV主要流行毒株97CN001的gp1452M或其免疫原性片段。
  7. 权利要求1-4任一项的重组复制型痘苗病毒，其中所述第二多核苷酸包含与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第二多核苷酸编码来自HIV-1的gp1452M。
  8. 权利要求1-7任一项的重组复制型痘苗病毒，其还包含编码额外的HIV抗原的第三多核苷酸。
  9. 权利要求8的重组复制型痘苗病毒，其中所述第三多核苷酸插入至HA区。
  10. 权利要求9的重组复制型痘苗病毒，其中所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag并包含SEQ ID NO:2所示的核苷酸序列。
  11. 权利要求9的重组复制型痘苗病毒，其中所述第三多核苷酸编码来源于中国HIV主要流行毒株97CN001的gag或其免疫原性片段。
  12. 权利要求9的重组复制型痘苗病毒，其中所述第三多核苷酸包含与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％、或至少99％相同性的核苷酸序列，且所述第三多核苷酸编码来自HIV-1的gag。
  13. 重组复制型痘苗病毒，其是天坛株痘苗病毒，其包含插入至天坛株痘苗病毒的TK区的编码来自中国HIV主要流行毒株97CN001的gp1452M的基因和编码HSV-TK的基因，并包含插入至天坛株痘苗病毒的HA区的编码97CN001的gag的基因。
  14. 活载体HIV疫苗组合物，其包含权利要求1-13中任一项的重组复制型痘苗病毒和药用可接受载体和/或佐剂。
  15. 权利要求1-13中任一项的重组复制型痘苗病毒在制备用于预防和/或治疗对象的HIV感染的活载体HIV疫苗中的用途。
  16. 权利要求15的用途，其中所述对象是免疫抑制患者。
  17. 权利要求16的用途，其中所述对象患有先天性免疫系统缺陷、HIV感染或正接受免疫抑制治疗。
  18. 试剂盒，其包含多个成份和指示免疫程序的说明书，其中一个成份为权利要求14的活载体HIV疫苗组合物。
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