WO2011032489A1 - Procédé d'utilisation d'un vaccin contre le vih à vecteur de poxvirus combiné à un vaccin contre le vih à vecteur adénoviral, et applications correspondantes - Google Patents

Procédé d'utilisation d'un vaccin contre le vih à vecteur de poxvirus combiné à un vaccin contre le vih à vecteur adénoviral, et applications correspondantes Download PDF

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WO2011032489A1
WO2011032489A1 PCT/CN2010/076905 CN2010076905W WO2011032489A1 WO 2011032489 A1 WO2011032489 A1 WO 2011032489A1 CN 2010076905 W CN2010076905 W CN 2010076905W WO 2011032489 A1 WO2011032489 A1 WO 2011032489A1
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hiv vaccine
vector
vaccine
poxvirus
vector hiv
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Chinese (zh)
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陈凌
陈志伟
张林琦
孙彩军
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中国科学院广州生物医院与健康研究院
港大科桥有限公司
清华大学
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    • 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
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
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    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biotechnology, and particularly relates to a novel method for preventing and treating AIDS by using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine.
  • AIDS Acquired Immune Deficiency Syndrome
  • WHO World Health Organization
  • UNAIDS announced at the end of 2008 that the total number of HIV-infected people worldwide is about 33 million, and the number of AIDS patients who have died has exceeded 30 million.
  • WHO World Health Organization
  • UNAIDS announced at the end of 2008 that the total number of HIV-infected people worldwide is about 33 million, and the number of AIDS patients who have died has exceeded 30 million.
  • 2007 alone there were 2.1 million people died of AIDS and 2.7 million were infected with HIV; Asia and Eastern Europe have become the fastest-growing areas of HIV (UNAIDS report, 2008).
  • the HIV epidemic trend in China is also quite serious.
  • HIV vaccines In the past one or two decades, although there have been many advances in the treatment of AIDS in drug treatment, the AIDS mortality rate in developed countries has dropped significantly, and some new drugs (such as antiretroviral drugs) that can treat and control HIV have emerged. , protease inhibitors, etc.) and new therapies (combined cocktails), but once the body is infected, it is impossible to eradicate the HIV virus, and due to high variability, the resistance of the HIV virus is becoming more serious, and drug treatment brings to HIV-infected people. The great adverse reactions and the affordability of patients make the prospects of these therapies unsatisfactory, so the development of effective HIV vaccines is still the most urgent need. In the field of HIV vaccines, it is generally believed that the study of HIV vaccines can be divided into three stages:
  • Phase 1 The AIDS vaccine study, which began in 1984, focused on the induction of antibodies to prevent viral infections, without considering the role of cellular immunity.
  • the second stage AIDS vaccine research emphasizes the role of cellular immune response.
  • This stage is mainly based on recombinant viral vector vaccines that induce cellular immune responses (vaccinia, adenovirus, canarypox virus, etc.), because theoretically, cellular immune responses can effectively control the replication and infection of HIV, and mathematical models also show that A reduction in viral load by 1 log can effectively reduce the rate of transmission of the population, which allows clinical trials to see the application prospects of this vaccine.
  • One of the most notable results is the AIDS vaccine developed by Merck, which is based on human adenovirus type 5, and the SHIV89.6P model has demonstrated a better immune response to CD8 T cells induced by HIV. Protection. Merck then pushed the vaccine into clinical trials and entered the clinical lib phase by 2004, but according to clinical trial results published in September 2007, the simple emphasis on cellular immune responses does not provide protection.
  • the current prevailing view, the third stage, is that an effective AIDS vaccine needs to induce a balanced humoral and cellular immune response.
  • the antibody acts as the first line of defense to neutralize part of the virus, which gives the subsequent cell-mediated memory response an activation time, while a strong cellular immune response removes the virus-infected cells and reduces the viral load. It can reduce the spread of HIV in the population.
  • McElrath et al. systematically analyzed Merck's HIV clinical trial data and found that although it did not To protect, but the vaccine produced a strong immune response in most of the tested population (77%), and the analysis also found that specific CD8+ T cells in these populations mainly secrete IFN- ⁇ cytokines alone. (73%), some also secrete TNF- ⁇ , but very few cells that secrete IL-2 cytokines or lymphocytes that secrete multiple cytokines at the same time.
  • the SIVmac251 virus in this group of monkeys The peak load is reduced by 1.4 log values, and the viral load at the setpoint is reduced by 2.4 log values. Moreover, during the whole experiment period (more than 500 days), the monkeys in this group did not die of AIDS or had obvious AIDS symptoms, but other groups of monkeys had different degrees of morbidity.
  • HIV-based vaccines should be able to induce a stronger, broader spectrum and versatility of T lymphocyte responses, while It induces a certain humoral immune response, which is likely to produce effective immune protection against HIV.
  • humoral immune response which is likely to produce effective immune protection against HIV.
  • researchers should experiment with a variety of different types of vaccines in combination to find the best combination.
  • Ad vectors have the advantages of high infection efficiency and high level of exogenous gene expression, high titer recombinant virus preparation, and large capacity. Therefore, Ad vectors are favored as mammalian cell expression vectors, recombinant vaccines and gene therapy vectors. At present, there are 342 clinical trials of infectious diseases, cancer, cardiovascular diseases, and single-gene diseases in the world using adenovirus as a carrier, and it ranks first among all kinds of carriers (24.8%).
  • VTT Tiantan strain poxvirus
  • the poxvirus vector used in this study was a further attenuated MVTT strain, and its experimental evidence showed that its neurotoxicity was greatly reduced. Therefore, it can be used as a safe smallpox vaccine or as a live carrier for other pathogen vaccines.
  • this vector can induce systemic mucosal reactions through mucosal pathways such as nasal and oral.
  • the present invention provides a method of using a combination of a poxvirus vector and an adenovirus vector, which can be used for the prevention and treatment of HIV and other infectious diseases and tumors.
  • the invention combines the use of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine, specifically using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine to induce a stronger and broader versatility.
  • the T lymphocyte reaction and the humoral immune response thereby effectively preventing and treating HIV infection.
  • the method can first use the poxvirus vector HIV vaccine to immunize the body, and then use the adenovirus vector HIV vaccine to immunize the body.
  • the method can also first use an adenovirus vector HIV vaccine to immunize the body, and then use a poxvirus vector HIV vaccine to immunize the body.
  • the poxvirus vector HIV vaccine contains the HIV gag, pol, env gene.
  • the poxvirus vector HIV vaccine further comprises a helper regulatory protein gene comprising the nef, vpx, vpr, vif, rev and tat genes.
  • the adenovirus vector HIV vaccine contains the HIVgag, pol, env gene.
  • the adenoviral vector HIV vaccine further comprises a helper regulatory protein gene comprising the nef, vpx, vpr, vif, rev and tat genes.
  • the means for immunizing the body means immunizing the body through a mucosal route, such as the nasal cavity, oral cavity, etc.; immunizing the body by intramuscular injection; by intravenous injection and all other routes of use of the vaccine.
  • a mucosal route such as the nasal cavity, oral cavity, etc.
  • the adenoviral vector described above refers to any adenovirus subtype known to those skilled in the art, an isolate or other animal derived adenovirus. Specifically, it may be a type 5 or type 2 adenoviral vector.
  • the poxvirus vector described above refers to any of the poxvirus subtypes known to those skilled in the art, and is detached. Specifically, it may be a modified Tiantan strain poxvirus vector.
  • the present invention also provides the above method for using a combination of a poxvirus vector and an adenovirus vector in the field of prophylactic and therapeutic HIV vaccines for preventing and treating AIDS, and for preventing and controlling hepatitis B, Ebola virus and tumors. .
  • a poxvirus vector carrying the HIV gag, pol, env gene and an adenoviral vector immunizes the body to induce a stronger, broader spectrum and versatility. T lymphocyte reaction, and showed strong protection and control of the rectal infection of SIVmac239 virus.
  • the strategy of enhancing the body to produce a more intense, linguistic and versatile T lymphocyte response is also suitable for the prevention and treatment of various viral infections, such as hepatitis B, Ebola, etc., as well as various tumors.
  • various viral infections such as hepatitis B, Ebola, etc.
  • poxvirus vectors and adenoviral vectors have been widely used in clinical trials, they have good safety, and the clinical application prospect of this strategy is very bright.
  • the invention is generally applicable to the prevention and treatment of AIDS, as well as the prevention and control of other infectious diseases and tumors.
  • Figure 1 is a graph showing the results of detection of recombinant adenoviral protein expression by western-blot method
  • Figure 2 is a comparison of immunogenicity of primary and boosted recombinant adenovirus in mice
  • Figure 2A is a recombinant adenovirus Comparison of immunogenicity after primary immunization in mice
  • Figure 2B is a comparison of immunogenicity of recombinant adenovirus after booster immunization in mice;
  • Figure 3 is a combination of a poxvirus vector (MVTT) SIV vaccine and an adenovirus (Ad5) vector SIV vaccine in a rhesus monkey immunization and challenge program;
  • MVTT poxvirus vector
  • Ad5 adenovirus
  • Figure 4 is the immunogenicity of the ELISPOT assay in combination with the MVTT vector and the Ad5 vector in rhesus monkeys;
  • Figure 4A is the ELISPOT test data from the last immunization for 6 weeks, and
  • Figure 4B is the ELISPOT test data from the last immunization for 21 weeks;
  • Figure 5 shows the results of detection of cytokine secretion by Gag peptide-stimulated CD8+ T cells by multicolor flow technique 6 weeks after booster immunization with MVTT and Ad5 vector HIV vaccine
  • Figure 6 shows the detection of cytokine secretion by memory T cells in peripheral blood by multicolor flow technique 16 weeks after MVTT combined with Ad5 vector HIV vaccine for booster immunization
  • Figure 6A shows the effect of detection.
  • Figure 6B is experimental data for detecting secretion of various cytokines by central CD8 T cells;
  • Figure 7 is a graph showing the proliferative ability of T lymphocytes stained with live cell dye carboxyfluorescein acetoacetate succinimidyl ester (CFSE) 16 weeks after booster immunization with MVTT and Ad5 vector HIV vaccine.
  • Figure 7A shows the proliferation of CD4 T cells;
  • Figure 7B shows the proliferation of CD8 T cells;
  • Figure 8 shows the cellular immune response against SIV individual antigens after SIVmac239 challenge;
  • Figure 8A is for structural proteins (Gag, The immune response of Pol and Env);
  • Figure 8B is the immune response to the helper regulatory proteins (Nef, Vpx, Vpr, Vif, Rev and Tat);
  • Figure 9 shows the level of SIV-specific antibody response before and after SIVmac239 infection by ELISA.
  • Figure 10 is a graph showing the results of data analysis of the peak viral load in experimental monkeys by quantitative PCR.
  • Example 1 Construction of adenovirus and poxvirus vaccine carrying SIVgag, pol and env genes and immunogenicity in mice
  • the individual genes required for the experiment were obtained by whole-genome synthesis.
  • This example relates to all three genes of the SIVmac239 virus, namely SIVmac239gag, pol and env.
  • the amino acid sequences of these proteins were obtained from the NCBI database and then reverse translated into DNA sequences according to human codons without any change in their amino acid sequence, allowing these antigens to be efficiently expressed in primate cells.
  • the above genes are separately introduced into an adenovirus vector, wherein the adenoviral vector is a human type 5 adenovirus lacking the E1 and E3 regions, and then rescued to obtain a recombinant adenovirus carrying the gene of interest, with a small amount of expansion.
  • the expression and genomic digestion were identified, and then amplified in Trex293 cells and purified by cesium chloride gradient density centrifugation. The purified virus was identified again and the infection titer TCID 5 was determined. And the concentration of the physical particles, and then the frozen storage and standby (refer to the applicant's patent, application number 200710026295.X).
  • Figure 1 shows the detection of recombinant adenovirus eggs by western-blot method.
  • the purified recombinant adenovirus can express the protein of interest at a high level, and these proteins can be further processed into various protein components in a natural form.
  • Ad5-gag can express P55 and P27
  • Ad5-pol can express P66/51, P31, P10, etc.
  • Ad5-env can express gpl 60, gpl20, gp41.
  • FIG. 1 is a comparison of immunogenicity of primary and boosted recombinant adenovirus in mice;
  • Figure 2 is a comparison of immunogenicity of primary recombinant immunized virus in mice;
  • Figure 2 is a recombinant gland Comparison of immunogenicity of the virus in mice after booster immunization; as shown in Fig.
  • mice immunized with SIVgag, pol and env simultaneously an immune response against the three SIV antigens was simultaneously produced.
  • This group has a reduced level of immune response compared to immunization of an antigen alone, which may be related to the high level of expression of multiple antigens in the body, leading to mutual competition between antigens.
  • the poxvirus vector MVTT-gpe (modified poxvirus Tiantan strain) expressing SIVgag, pol and env was constructed and provided by Professor Chen Zhiwei of the Institute of AIDS, Li Ka Shing Medical College, University of Hong Kong, and their expression and antigenicity were confirmed by them ( Huang X et al. Vaccine. 2007).
  • Example 2 Combination of poxvirus vector SIV vaccine and adenovirus vector Immunization of SIV vaccine in rhesus monkey
  • the vaccinia vector MVTT-gpe expressing SIVgag, pol and env was constructed and provided by Professor Chen Zhiwei of the AIDS Research Institute of the Li Ka Shing Faculty of Medicine, University of Hong Kong; the recombinant adenoviruses (S5g, pol and env expressed above) (Ad5-SIVgag, Ad5-SIVpol and Ad5-SIVenv).
  • Monoclonal antibodies used (anti-human CD3-pacific blue, anti-NHP CD4-FITC, anti-human CD4-AmCyan, anti-human CD8-PerCP, anti-human CD8-APC-Cy7 (SKI), anti-human CD28-FITC , anti-human CD95-PE-Cy5, anti-human TNFa-PE-CY7, anti-human IFNy-PE, anti-human IL2-APC) were purchased from BD.
  • peptides are from the National Institutes of Health AIDS Research and Reference Reagent Program (NIH AIDS) The Research & Reference Reagent Program provides that most peptides consist of 15 amino acids with 11 amino acid overlaps between each other. Purity >80%.
  • the solution was dissolved in dimercaptosulfoxide (DMSO) to prepare a solution of 0.4 mg/ml/peptide, and stored at -70 ° C after dispensing.
  • DMSO dimercaptosulfoxide
  • ELISPOT board brand: Millipore; article number: MSIPS4510
  • SIV virus particle lysate was prepared for itself
  • TMB/E substrate catalog: Chemicon, USA.
  • BCIP/NBT substrate brand: Pierce; article number: 34042
  • Cell dye CFSE was purchased from Molecule Probe.
  • Streptomycin-conjugated alkaline phosphatase brand: BD PharMingen, Cat. No. 554065 was purchased from Gene Co., Ltd.
  • MVTT poxvirus vector
  • Ad5 adenovirus
  • Detection indicators include ELISPOT, multifunctional T cells, cell proliferation and antibody titers.
  • the adenovirus was immunized by intramuscular injection at a dose of 10" vp/monkey.
  • the amount of poxvirus was lxlO 8 PFU/ml/monkey, nasal immunization and sublingual immunization.
  • the isolated PBMC cells were counted and adjusted to 2 ⁇ 10 6 /ml, and added to a 24-well culture plate at 1 mL per well.
  • the medium is R10.
  • test antibody biotinylated anti-monkey IFN- ⁇ antibody
  • PBST containing 5 % FBS, ⁇ /well, overnight at 4 °C.
  • Streptomycin-conjugated alkaline phosphatase was diluted 1:2500 with PBST containing 5 % FBS, ⁇ /well, 37 ° C, for 2 hours.
  • BCIP/NBT substrate (Pierce, Cat: 34042), warm bath at 37 °C for 30 min.
  • the vacuum pump pumps off the supernatant. Oscillating on a vortex shaker for 2 - 3 seconds.
  • Multicolor flow cytometry data was analyzed using FlowJo software. Statistical analysis and mapping were performed using JMP version 6.0.3 software. Differences between groups were compared using a nonparametric Wilcoxon rank test.
  • FIG. 4 shows the immunogenicity of the ELISPOT assay in combination with the MVTT vector and the Ad5 vector in rhesus monkeys;
  • Figure 4A shows ELISPOT data from the last immunization for 6 weeks, and
  • Figure 4B shows ELISPOT data from the last immunization for 21 weeks.
  • MVTT modified vaccinia virus Tiantan strain
  • IFN- ⁇ ELISPOT technology has become a relatively versatile and widely accepted indicator for evaluating HIV vaccines, today's more recognized view is that immunoprotection and the number of lymphocytes with multiple functions are more relevant, so-called multi-functional Lymphocytes are lymphocytes that simultaneously secrete various cytokines including IFN- ⁇ , including IL-2, TNFa, MIP 1 ⁇ and the like. Evaluation of multifunctional sputum lymphocytes will gradually become an important indicator for evaluating the effectiveness of HIV vaccines. To this end, we have developed and improved multi-color flow technology to determine the versatile sputum lymphocyte response produced by this strategy.
  • Fig. 5 shows the results of detection of cytokine secretion by Gag peptide-stimulated CD8+ T cells by multicolor flow technique 6 weeks after MVTT and Ad5 vectors were combined with rhesus monkeys; After 6 weeks of immunization with the Ad5 vector, the monkeys in the combined immunization group not only greatly enhanced the ability to secrete a certain cytokine alone, but more importantly, detected more simultaneous secretion of IFN-y/TNF-a/IL-2 cells.
  • Factor of multifunctional CD8+ T lymphocytes are examples of multifunctional CD8+ T lymphocytes.
  • CD8+ T lymphocytes secrete three cytokines of IFN- ⁇ /TNF-a/IL-2, 1.4668 ⁇ 1.1845% of CD8+ T lymphocytes secrete both IFN- ⁇ /TNF-a cytokines; while only 0.0320 ⁇ 0.0472% of CD8+ T lymphocytes secrete IFN- ⁇ /TNF-a /IL-2 in monkeys immunized with Ad5 vector alone.
  • MVTT combined with Ad5 vector produced 7.7 times more multifunctional CD8+ T lymphocytes that secrete IFN- ⁇ /TNF-a/IL-2 simultaneously. More than 9 times more multifunctional CD8+ T lymphocytes capable of secreting both cytokines of IFN- ⁇ /TNF-a.
  • Fig. 6 is a test result of detecting the secretion of cytokines by memory T cells in peripheral blood by multicolor flow technique 16 weeks after the combination of MVTT and Ad5 vector for boosting rhesus monkeys; 6A is experimental data for detecting secretion of various cytokines by effector CD8 T cells; and FIG. 6B is experimental data for detecting secretion of various cytokines by central CD8 T cells.
  • SIV antigen-specific memory CD4+ T cells and CD8+ T cells to produce and secrete cytokines is significantly stronger in these monkeys.
  • the effector CD8 + cells in peripheral blood of monkeys in the MVTT and Ad5 combined immunization groups were significantly more potent in secreting cytokines. These cells secreted IFN-Y/TNF-a cytokines simultaneously. the Lord. Although the ability of central memory CD8+ cells to secrete cytokines was not significantly different in each group of experimental monkeys, these cells were significantly more potent in secreting IL-2 than in effector memory T cells. In conclusion, we detected that MVTT and Ad5 co-immunization can induce more multifunctional memory T lymphocytes in rhesus monkeys. Further analysis showed that this method induced the simultaneous production of central memory and effector memory T cells, with the effect memory CD8 + T fine packets.
  • Fig. 7 is a graph showing the results of detecting the proliferative ability of T lymphocytes by CFSE staining after 16 weeks of booster immunization with MVTT and Ad5 vector;
  • Fig. 7A shows the proliferation of CD4 T cells;
  • Fig. 7B shows the CD8 T cells.
  • the proliferation situation As shown in Figure 7A, MVTT and Ad5 vector combined with monkey-derived PBMC were used in vitro. After SIV Gag stimulation, (4.2725 ⁇ 4.5516)% of CD4+ cells were CFSElow, ie, proliferating progeny cells; whereas the AdB vector-immunized monkey PBMCs were stimulated with SIV Gag (3.2350 ⁇ 1.7008)% of CD4+ T cells. CFSElow.
  • CD8+ T cells As shown in Fig. 7B, after stimulation with SIV Gag, there were (7.4825 ⁇ 5.8550 %) CD8+ T cells as CFSElow, which is a proliferating progeny cell; whereas the Ad5 vector alone in the wolf PBMC was (2.9475 ⁇ 1.6951) %. The CD8+ T cells are CFSElow.
  • the proliferation of CD8+ T lymphocytes against antigen-stimulated MVTT was significantly increased by 2.5-fold compared with the Ad5 vector.
  • the MVTT vector priming the Ad5 vector-enhanced immunization method produced a stronger, broader spectrum and more functional T cell response in Chinese rhesus monkeys, which is in line with the recent evaluation of effective vaccine indicators. This strategy deserves further study.
  • Example 3 Combined use of poxvirus vector and adenovirus vector Immunoprotection test of SIV vaccine in rhesus monkey
  • the SIVmac239 virus was obtained by the inventor, and the SIVmac239 phage molecular clone used was provided by the National Institutes of Health AIDS Research and Reference Reagent Program.
  • Monoclonal antibody used (anti-human CD3 -pacific blue, anti-NHP CD4-FITC, anti-human CD4-AmCyan, anti-human CD8-PerCP, anti-human CD8-APC-Cy7 (SKI), anti-human CD28-FITC, anti-human CD95-PE-Cy5, anti-human TNF (x-PE-CY7, anti-human IFNy-PE, anti-human IL2-APC) was purchased from BD.
  • HRP-anti-monkey IgG was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.
  • Quantitative RT-PCR Kit Quantitative RT-PCR Kit (QuantiTect SYBR Green RT-PCR Kit, Cat. No.: 204243) purchased from Qiagen, FACS lysis solution, BD TruCOU T Tubes, Cat. No. 340334 BD Biosciences. See Example 2 for other reagents.
  • the immunization schedule of each group is shown in Fig. 3.
  • the SIVmac239 virus was infected with rectal infection for 22 weeks at the last immunization, and the dose was 10 5 TCID 5 .
  • the specific method is that the monkey is fasted for 24 hours, keep the hip high, insert the anus 4 _ 7cm into the anus with a stomach tube, enter the virus, and inject a small amount of air. Keep this position for at least 20 minutes.
  • the kit comes with primer 2 ( ⁇ ) 0.5 ⁇ 1
  • the virus copy number of each sample was obtained by Opticon Monitor 3 software, and then the virus copy number per ml of blood was obtained according to the dilution factor.
  • Blocking PBST containing 5% non-fat dry milk powder, 250 ⁇ l per well, blocked at 37 ° C for 1 h.
  • Figure 8 is a cellular immune response against SIV individual antigens after challenge with SIVmac239;
  • Figure 8A is an immune response against structural proteins (Gag, Pol and Env);
  • Figure 8B is for helper regulatory proteins (Nef, Vpx, Vpr , Vif, Rev and Tat) immune response.
  • the monkeys in the combined immunization group rapidly increased the response to the vaccine-carrying immunogen (gag/pol/env), while the control group responded slowly and reacted.
  • the intensity is also relatively weak. This indicates the immunity of the present invention
  • the method effectively produces memory T cells that are rapidly activated upon stimulation with the same antigen and produce a corresponding immune response.
  • the response to the immunogen (nef/vpx/vpr/vif/rev/tat) that was not carried by the vaccine in the monkeys of the co-immunized group was significantly weaker than that of the control group.
  • the body is effectively controlled at a relatively low level or is not infected, so that the level of response to the antigen carried by the virus itself is relatively low. This guess was confirmed in the next viral load assay.

Abstract

L'invention concerne un procédé visant à immuniser des sujets à l'aide d'un vaccin contre le VIH à vecteur de poxvirus combiné à un vaccin contre le VIH à vecteur adénoviral, ce procédé pouvant servir à prévenir ou à traiter le SIDA et d'autres maladies et tumeurs infectieuses.
PCT/CN2010/076905 2009-09-15 2010-09-14 Procédé d'utilisation d'un vaccin contre le vih à vecteur de poxvirus combiné à un vaccin contre le vih à vecteur adénoviral, et applications correspondantes WO2011032489A1 (fr)

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CN101670102A (zh) * 2009-09-15 2010-03-17 中国科学院广州生物医药与健康研究院 联合使用痘病毒载体hiv疫苗与腺病毒载体hiv疫苗的方法及其应用
CN102258779B (zh) * 2011-07-18 2013-07-24 曾毅 四种及四种以上hiv载体基因疫苗序贯及重复应用
CA2960096A1 (fr) * 2014-09-03 2016-03-10 Bavarian Nordic A/S Procedes et compositions pour augmenter des reponses immunitaires
CN110624106A (zh) * 2018-06-22 2019-12-31 嘉和生物药业有限公司 Pd-1信号通路拮抗剂与艾滋病疫苗的联合应用

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CN101219221A (zh) * 2007-01-12 2008-07-16 中国科学院广州生物医药与健康研究院 携带hiv基因的腺病毒载体疫苗
CN101394868A (zh) * 2006-03-07 2009-03-25 浙江海正药业股份有限公司 重组腺病毒5型/35型和痘病毒mva活病毒载体疫苗的联用诱导强大的免疫应答的方法
CN101670102A (zh) * 2009-09-15 2010-03-17 中国科学院广州生物医药与健康研究院 联合使用痘病毒载体hiv疫苗与腺病毒载体hiv疫苗的方法及其应用

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CN1695737A (zh) * 2005-05-20 2005-11-16 吉林大学 冻干剂型艾滋病重组痘病毒安卡拉株载体疫苗及其制备方法
CN101394868A (zh) * 2006-03-07 2009-03-25 浙江海正药业股份有限公司 重组腺病毒5型/35型和痘病毒mva活病毒载体疫苗的联用诱导强大的免疫应答的方法
CN101219221A (zh) * 2007-01-12 2008-07-16 中国科学院广州生物医药与健康研究院 携带hiv基因的腺病毒载体疫苗
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