WO2012139281A1 - Adjuvant à base de protéine tat du vih et ses utilisations - Google Patents

Adjuvant à base de protéine tat du vih et ses utilisations Download PDF

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WO2012139281A1
WO2012139281A1 PCT/CN2011/072665 CN2011072665W WO2012139281A1 WO 2012139281 A1 WO2012139281 A1 WO 2012139281A1 CN 2011072665 W CN2011072665 W CN 2011072665W WO 2012139281 A1 WO2012139281 A1 WO 2012139281A1
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vaccine
hiv
protein
fragment
variant
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PCT/CN2011/072665
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Chinese (zh)
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邵一鸣
刘野
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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 PCT/CN2011/072665 priority Critical patent/WO2012139281A1/fr
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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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides

Definitions

  • the present invention relates to the field of adjuvants, and methods and uses of adjuvants for modulating immune responses induced by a vaccine of interest.
  • the invention particularly relates to an adjuvant comprising a HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof, and a method and use of the adjuvant to enhance an immune response induced by a vaccine of interest .
  • DNA vaccines are techniques that use genetically engineered DNA molecules to induce an immune response in an organism to protect the organism from infection by viruses, bacteria, and the like.
  • One of the advantages of DNA vaccine technology is that it induces various types of immune responses.
  • clinical trials of DNA vaccines have provided exciting safety data.
  • DNA vaccines are well tolerated by the body in preclinical and clinical trials, demonstrating good safety ( See, for example, Sandhya Vasan, Sarah J. Schlesinger. et. al. Phase 1 Safety and Immunogenicity Evaluation of AD VAX, a Multigenic, DNA-Based Clade C/B, HIV-1 Candidate Vaccine, PLoS One. 2010; 5(1) : e8617 and Thomas C. Greenough, Coleen K.
  • DNA vaccines are still in the experimental phase and there are not many clinical applications. A very important reason is that DNA vaccines are still proven to be less immunogenic in human experiments. As a result, DNA vaccines are used in large doses, and the number of immunizations is high or must be combined with other vaccines, which increases the cost of production and use of vaccines and greatly limits the application of DNA vaccines.
  • the ability to significantly increase the immunogenicity of DNA vaccines is a critical factor in determining the future of DNA vaccine technology (Lu S, Wang S, Grimes-Serrano JM. Current progress of DNA vaccine studies in humans. Expert Rev Vaccines. 2008 Mar; 7(2): 175-91).
  • adjuvant is one of the effective strategies to improve the immunogenicity of DNA vaccines.
  • people gradually realize the immunity induced by direct injection of naked DNA for immunization.
  • Both response and immune memory are not ideal, but a stronger, more durable immune effect can be achieved by combining with an adjuvant (Barouch DH, Santra S, Schmitz JE, Kuroda MJ, et al. Control of viremia and prevention of clinical AIDS in rhesus Monkeys by cytokine-augmented DNA vaccination . Science, 2000, 290(5491): 486-92; Min W, Lillehoj HS, Bumside J, et al.
  • An immunological adjuvant refers to a substance which is applied to the same subject simultaneously or separately, and which can non-specifically enhance or change the body's specific immune response against the antigen.
  • the adjuvant itself may or may not be antigenic, but the adjuvant must be safe according to the recommendations of the World Health Organization.
  • HIV Tat is a transcriptional transactivator of human immunodeficiency virus whose primary function is to regulate the transcription of HIV and promote the transcription of HIV genes by binding to the LTR region of the HIV genome.
  • HIV Tat protein can promote the activation and maturation of DC cells, thereby enhancing the antigen presentation ability of the body and effectively improving the immune response level of the body (Emanude Fanales-Belasio, Sonia Moretti, Filomena Nappi, et al. Native HIV-1 Tat Protein Targets Monocyte-Derived Dendritic Cells and Enhances Their Maturation, Function, and Antigen-Specific T Cell Responses.
  • the present invention is the first to use HIV Tat as an adjuvant in combination with a vaccine, and has confirmed its enhancement and regulation effect on the immune response induced by the vaccine in an animal model. Summary of invention
  • One aspect of the invention relates to the use of an adjuvant for enhancing an immune response induced by a vaccine of interest in a subject.
  • the invention relates to HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding HIV Tat protein or a fragment or variant thereof, for the preparation of an immune response induced in a subject for enhancing a vaccine of interest in a subject Use in a pharmaceutical or vaccine composition.
  • the vaccine of the invention comprises a DNA vaccine
  • the adjuvant or vaccine composition comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • the vaccine of the invention comprises a protein vaccine
  • the adjuvant or vaccine composition comprises HIV Tat protein or a fragment or variant thereof.
  • the vaccine of interest of the invention comprises an HIV Env protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine of interest of the invention comprises an HIV encoding Polynucleotides of Env proteins, and/or HIV Gag proteins, and/or HIV Pol proteins, or fragments or variants thereof.
  • the invention also relates to a method of enhancing an immune response induced by a vaccine of interest in a subject.
  • the method of the invention comprises administering the vaccine to a subject, and administering to the subject a HIV Tat protein or fragment or variant thereof or encoding an HIV Tat protein prior to, concurrently with, or after administration of the vaccine.
  • the vaccine of the method of the invention comprises a DNA vaccine
  • the adjuvant comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof as described in the methods of the invention is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • the vaccine of the method of the invention comprises a protein vaccine, and the adjuvant comprises HIV Tat protein or a fragment or variant thereof.
  • the vaccine in the methods of the invention comprises an HIV Env protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine comprises an HIV Env protein encoding And/or a polynucleotide of HIV Gag protein, and/or HIV Pol protein, or a fragment or variant thereof.
  • the vaccines and/or adjuvants of the invention are administered by a muscle and/or intradermal route of administration.
  • the invention also relates to a vaccine composition.
  • the vaccine composition of the invention comprises the following components: (a) a vaccine, A polynucleotide comprising an immunogen and/or encoding an immunogen; and (b) an adjuvant comprising an HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the vaccine in a vaccine composition of the invention comprises a DNA vaccine
  • the adjuvant comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • the vaccine in the vaccine composition of the invention is a protein vaccine, and the adjuvant comprises HIV Tat protein or a fragment or variant thereof.
  • the vaccine in a vaccine composition of the invention comprises an HIV Env protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine of interest comprises a coding A polynucleotide of HIV Env protein, and/or HIV Gag protein, and/or HIV Pol protein, or a fragment or variant thereof.
  • the invention also relates to an adjuvant.
  • an adjuvant of the invention comprises a HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding a HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudovirus particle, and a virus-like particle.
  • Figure 1 Schematic diagram of mouse immunization schedule and time of detection. The mice were immunized once every 0 and 4 weeks, and the mice were subjected to blood collection to separate the serum from the eyelids at 7 weeks (3 weeks after the 2-needle immunization), and the spleen lymphocytes were isolated after the mice were sacrificed.
  • Figure 2 ELISA results of Env-specific binding antibodies in serum of mice 3 weeks after 2-needle immunization. To avoid interference with the graph, the error bars are omitted.
  • the ELISA results of the mice after 3 weeks of immunization showed that the DNA vaccine plus adjuvant group significantly increased the intensity of the vaccine-induced Env-specific binding antibody compared with the DNA alone immunization group.
  • Figure 3 Specific cellular immunity results of HIV-1 Env, Gag, Pol were detected by ELISPOT assay 3 weeks after 2-needle immunization. The results of cellular immunoassay were expressed by the number of cells secreting IFN- ⁇ after stimulation of mouse spleen cells with Env, Gag, and Pol specific peptides, respectively. ELISPOT detection of mouse IFN- ⁇ The results showed that the DNA vaccine plus adjuvant group significantly increased the level of vaccine-induced cellular immune response (PO.01) compared to the DNA alone immunization group.
  • PO.01 vaccine-induced cellular immune response
  • One aspect of the invention relates to the use of an adjuvant for enhancing an immune response induced by a vaccine of interest in a subject.
  • the invention relates to HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding HIV Tat protein or a fragment or variant thereof, for the preparation of an immune response induced in a subject for enhancing a vaccine of interest in a subject Use in a pharmaceutical or vaccine composition.
  • vaccine and “target vaccine” as used herein are used interchangeably and refer to: a class of organisms that prevent or treat infectious diseases in order to effectively prevent and control the occurrence, spread, and inoculation of infectious diseases. product. It can be classified into various types such as, but not limited to, DNA vaccines, protein vaccines, viral vector vaccines, attenuated vaccines, inactivated vaccines, and the like.
  • the terms “vaccine” and “vaccine of interest” may include an antigen or a polynucleotide encoding an antigen.
  • the antigen refers to an antigen that can induce an immune response in a subject, the immune response involving the alleviation, suppression, alleviation and/or treatment of certain diseases, and the disease mainly refers to an infectious disease involving certain viruses.
  • infectious diseases such as malaria.
  • the vaccines described herein may include HIV-related vaccines, as well as vaccines for infectious diseases such as hepatitis A, hepatitis B, hepatitis C virus vaccine, human papilloma virus vaccine, yellow fever virus vaccine, and malaria vaccine.
  • vaccine composition means a composition containing a vaccine, and may further contain an auxiliary ingredient such as a pharmaceutically acceptable carrier, adjuvant or the like.
  • the term "adjuvant” refers to an immunomodulatory agent that is not itself immunogenic or has some immunogenicity, but can be used to stimulate the immune system to improve response to other immunogenic substances.
  • the antigen-induced immune response or the type of immune response of the antigen can be directly or indirectly modulated.
  • the immune response includes, for example, humoral immunity, cellular immunity, and the like.
  • HIV Tat refers to a transcriptional transactivator of human immunodeficiency virus, and herein the HIV Tat protein can include its full length protein or a functional fragment or variant of the protein.
  • fragment and “functional fragment” are used interchangeably and are meant to refer to portions of a protein that achieve the objectives of the invention.
  • variant and “functional variant” are also used interchangeably and are meant to refer to a mutant of a protein that achieves the objectives of the invention, for example by using one or more insertions, deletions, or substitutions. A mutant obtained by a method such as amino acid.
  • the vaccine of the invention comprises a DNA vaccine
  • the adjuvant or vaccine composition comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • DNA vaccine may also be referred to as a gene vaccine or a nucleotide vaccine or the like, which means that an expression vector containing a nucleotide sequence encoding an antigen is directly injected into an animal to make the nucleotide sequence in vivo. The resulting antigen thus activates the body's immune system, which can be used to induce specific humoral and cellular immune responses.
  • humoral immune response "cell immune response” as used herein has the meanings well known in the art.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • a vector as described herein refers to a system that is capable of carrying a foreign antigen and capable of being rendered in a host cell. These include plasmid vectors, recombinant viral vectors, recombinant bacterial vectors, pseudovirions, virus-like particles, and the like.
  • the "plasmid” refers to a bacterial plasmid vector, including various commercial and laboratory-constructed plasmid vectors, such as but not limited to pDRVISVl.O (DNA constructed by the Chinese Medicine Center for Disease Control and Prevention, Center for Disease Control and Prevention, China) For vaccine vectors, see CN 1560259 A (Chinese Patent Application 200410028280.3)).
  • the "recombinant viral vector” refers to a vector capable of carrying a foreign gene based on a recombinant virus, and includes, for example, a recombinant adenovirus vector, a recombinant poxvirus vector, a baculovirus vector, and the like.
  • the "recombinant bacterial vector” refers to a vector capable of carrying a foreign gene constructed based on a recombinant bacterium, and includes, for example, a Listeria vector, an attenuated Salmonella vector, and the like.
  • the "pseudovirus particle” refers to a virus having a single round of infection activity but not having a subsequent infection ability, which is produced by co-transfecting cells with HIV framework plasmid DNA and plasmid DNA carrying a membrane gene, including, for example, HIV pseudovirus.
  • the "viral-like particle” refers to a hollow particle which is one or more structural proteins containing a certain virus, which has no viral nucleic acid, cannot be autonomously replicated, and is identical or similar in morphology to a true virion, including, for example, an HIV virus. Sample particles, influenza virus-like particles.
  • insertion refers to the introduction of an antigen of interest into a vector of interest using techniques conventional in the art, such as the use of recombinant DNA techniques and the like.
  • the vaccine of the invention comprises a protein vaccine
  • the adjuvant or vaccine composition comprises HIV Tat protein or a fragment or variant thereof.
  • the vaccine of interest of the invention comprises an HIV Env protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine of interest of the invention comprises an HIV encoding Polynucleotides of Env proteins, and/or HIV Gag proteins, and/or HIV Pol proteins, or fragments or variants thereof.
  • HIV Env protein refers to the HIV envelope and its functional fragments or variants such as, but not limited to, the extramembranous portion of the HIV envelope protein, such as gpl45; the HIV Gag protein described herein is Refers to the core protein or coat protein encoded by the HIV gene and its functional fragments or variants, such as but not limited to P55; HIV Pol protein as used herein refers to various enzyme proteins and fragments thereof encoded by the HIV pol gene. Or variants such as, but not limited to, reverse transcriptase, integrase, or protease, and the like.
  • Modulation of a vaccine-induced immune response as described herein includes directly or indirectly promoting, accelerating, augmenting, or prolonging the immune response, such as humoral and/or cellular immunity; or altering the type of immune response, such as innate Immunity or acquired immunity.
  • the invention also relates to a method of enhancing an immune response induced by a vaccine of interest in a subject.
  • the method of the invention comprises administering the vaccine to a subject, and administering to the subject a HIV Tat protein or fragment or variant thereof or encoding an HIV Tat protein prior to, concurrently with, or after administration of the vaccine.
  • the adjuvant of the present invention may be used together with the vaccine or separately. Where used together can include applying the two directly together to apply to the subject. While being used alone may include pre-administering the adjuvant to a subject, and then administering the vaccine to the same subject. Alternatively, the adjuvant may be administered to the same subject before the vaccine is administered.
  • the time interval for administration is, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks.
  • the dosage range of the adjuvant used therein may, for example, Yes: From lg to 100 ⁇ ⁇ .
  • the dose as an example may be, for example, lg, 5 ⁇ g, 10 ⁇ ⁇ , 25 ⁇ g, 50 ⁇ g, or 100 ⁇ 8 . It should be understood that these choices are within the skill of the art and are within the skill of the art. The technical solution of the present invention therefore covers these options.
  • immunizing a subject can be carried out by any method known in the art.
  • the subject for immunization of the present invention may be a human or an animal commonly used in the art, such as, but not limited to, a mouse, a rat, a monkey, a rabbit, a cotton sheep, a goat, a horse, a cow, and the like.
  • the vaccine of the method of the invention comprises a DNA vaccine
  • the adjuvant comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof as described in the methods of the invention is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • the vaccine of the method of the invention comprises a protein vaccine, and the adjuvant comprises HIV Tat protein or a fragment or variant thereof.
  • the vaccine in the methods of the invention comprises an HIV ⁇ protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine comprises an HIV Env protein encoding And/or a polynucleotide of HIV Gag protein, and/or HIV Pol protein, or a fragment or variant thereof.
  • the vaccines and/or adjuvants of the invention are administered by a muscle and/or intradermal route of administration.
  • the invention also relates to a vaccine composition.
  • the vaccine composition of the invention comprises the following components: (a) a vaccine comprising an immunogen and/or a polynucleotide encoding an immunogen; and (b) comprising an HIV Tat protein or fragment thereof or A variant or an adjuvant encoding a polynucleotide of HIV Tat protein or a fragment or variant thereof.
  • the vaccine in a vaccine composition of the invention comprises a DNA vaccine
  • the adjuvant comprises a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding the HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudoviral particle, and a virus-like particle.
  • the vaccine in a vaccine composition of the invention is a protein vaccine, and the adjuvant comprises an HIV Tat protein or a fragment or variant thereof.
  • the vaccine in a vaccine composition of the invention comprises an HIV Env protein, and/or an HIV Gag protein, and/or an HIV Pol protein, or a fragment or variant thereof; or the vaccine of interest comprises a coding A polynucleotide of HIV Env protein, and/or HIV Gag protein, and/or HIV Pol protein, or a fragment or variant thereof.
  • the invention also relates to an adjuvant.
  • an adjuvant of the invention comprises a HIV Tat protein or a fragment or variant thereof or a polynucleotide encoding an HIV Tat protein or a fragment or variant thereof.
  • the polynucleotide encoding a HIV Tat protein or a fragment or variant thereof is inserted into a vector.
  • the vector is selected from the group consisting of a plasmid, a recombinant viral vector, a recombinant bacterial vector, a pseudovirus particle, and a virus-like particle. The invention is further illustrated below in conjunction with specific embodiments.
  • pDRVISVl.O is a DNA vaccine vector constructed by the Virus Immunology Room of the Center for STD/AIDS Prevention and Control of the Chinese Center for Disease Control and Prevention.
  • the vector pDRVISVl.O can be constructed in accordance with the method described in CN 1560259 A (Chinese Patent Application No. 200410028280.3), which is incorporated herein by reference.
  • a vaccine plasmid for the HIV BVC recombinant strain Env, Gag, Pol gene constructed on the basis of the DNA vaccine vector pDRVISV1.O.
  • Endofree Plasmid Giga Kit for the preparation of endotoxin-free plasmid DNA is a Qiagen product; the Mouse lFN-y ELISPOT assay kit is a product of U-CyTech.
  • Cell culture fluid was purchased from Hyclone Corporation.
  • Fetal bovine serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.
  • Anti-HIV-1 positive serum was obtained from the Reference Room of the STD/AIDS Prevention and Control Center (available through standard procedures); horseradish peroxidase HRP-labeled goat anti-mouse IgG was purchased from Beijing Zhongshan Company; derived from BVC recombinant subtype HIV-1 Synthetic peptide of the strain Env for ELISPOT analysis, by the National Institutes of Health The NIH AIDS Research & Reference Reagent Program is a gift (available free of charge); the HIV-1 Env antigen used in the ELISA test is provided by the HIV/AIDS Room of the STD HIV/AIDS Prevention and Control Center, which is based on conventional techniques.
  • plasmids used in this example were pDRVISVl.O, abbreviated as pl.0.
  • the HIV Tat sequence was inserted into the DRVISVl.O vector using the following primers:
  • Rv-Tat GAAGATCTTTAGTCGAATGGGTCTGTCTCTG (SEQ ID NO: 2)
  • the template used for the Tat sequence was synthesized by Invitra en, and its specific sequence is:
  • PCR PCR instrument purchased from Takara
  • step (f) Repeat step (e) - times.
  • the DNA was eluted by centrifugation at 14,000 rpm for 2 min.
  • the gel recovery gene fragment and the expression plasmid vector are separately digested, and the system is as follows:
  • connection system is as follows:
  • the transformed cells were separately applied to an LB agar plate containing kanamycin, and cultured at 37 ° C for 16 hrs or more.
  • Plasmid extraction (kits were purchased from Omega:). The plasmid was named: pl.0-Tat. 7. The final plasmid was sent to Invitrogen for sequencing.
  • the various DNA vaccines used in the examples of the present invention can be constructed by conventional techniques with reference to the above process.
  • the resulting plasmids were named pl. 0-Gag, pl. O-Env, and pl. 0-Pol, respectively.
  • the naming method of "plasmid name + target gene name" is used uniformly.
  • the following template is used:
  • Env template plasmid pDRVI3.1-env with the accession number CGMCC No. 2112 (see Chinese Patent Application No. 200710129786.7, publication No. CN101353665A, incorporated herein by reference).
  • Pol template plasmid pCRScript-gpnef with the accession number CGMCC No. 1003 (see Chinese Patent Application No. 200710088735.4, Publication No. CN 101270363A, the disclosure of which is incorporated herein by reference).
  • Gag template plasmid pCRScript-gpnef with accession number CGMCC No.1003 (see National Patent Application No. 200710088735.4, Publication No. CN 101270363A, the disclosure of which is incorporated herein by reference.
  • the primers used therein are:
  • Fwd-Gag ACGCGTCGACGCCACCATGGCCGCCAGGGCCAG (SEQ ID NO: 4)
  • Rv-Gag CGGGATCCTCACTGGCTGCTGGGGTCGTTG (SEQ ID NO: 5)
  • Fwd-Pol ACGCGTCGACGCCACCATGGCCGCCAGGGCCAGC (SEQ ID NO: 6)
  • Rv-Pol GATATCTTACTTGGTCCTGTGCTGGCCGATC (SEQ ID NO: 7)
  • Rv-Env GATATCTCAGTAGCCCTGCCTCACCCTG (SEQ ID NO: 9)
  • the annealing temperature was 60 ° C and extended at 72 ° C for 4 minutes.
  • mice 42 6-8 week old Balb/c female mice were divided into 6 immunized groups (three DNA vaccines each containing a single administration group and one additional adjuvant group), and one empty vector control group.
  • the grouping and immunization programs are shown in Table 1:
  • Immunization method For the DNA vaccine plus adjuvant immunization group, take 5 ( ⁇ L concentration of lg / L DNA vaccine and 5 ( ⁇ L concentration of lg / L pl. O-Tat mixed, then inject the mouse. For none In the DNA vaccine immunization group of adjuvant, 5 ( ⁇ L concentration of lg/L DNA and 50 ⁇ lg/L empty vector plasmid were mixed and injected into the mice. The empty vector group was taken as 5 ( ⁇ L concentration was The lg/L pDRVI1.O empty plasmid vector and 5 (L concentration of lg/L DNA Tat were mixed, and then injected into the mouse. The injection site was Balb/C mouse tibialis anterior muscle, and the left and right legs were each injected with 50 ⁇ M.
  • the immunization program and test time are shown in Figure 1: The immunization program is immunized once every 0 and 4 weeks. At the 7th week, the rats were anesthetized with barbital, and the blood was separated by eyelid collection. After the mice were sacrificed, the spleen lymphocytes were isolated.
  • Example 3 Humoral immunization with HIV Tat and Env antigen DNA vaccine The mice were immunized once every 0 and 4 weeks, and the rats were anesthetized with barbital after the 7th week, and the serum was separated by eyelid extraction. Env was detected by ELISA. The antigen) specifically binds to the antibody titer. The Env antigen protein was obtained by expressing the plasmid pl. O-Env for immunizing mice constructed above in a 293 suspension cell expression system (purchased from Invitrogen).
  • Step 1 Recombinant Env antigen protein coated ELISA plate (O.l g/ml) with HIV-1 BVC recombinant strain with a purity greater than 95%, and 0.1 ml of the corresponding protein antigen per well, overnight at 4 °C. The next day, the washing buffer was washed 3 times, and the residual liquid was drained. The antibody was diluted with antibody dilution for 60 min, washed with buffer for 3 times, dried for testing, or dried at 4 ° C;
  • Step 2 Serially dilute the antibody dilution to the sample to be tested (diluted sample is mouse serum:) (starting at JA 1:100, diluted to 1: 12800) 0.1 ml added to the above coated reaction well Incubate at 37 ° C for 60 min, wash 8 times (while doing blank, negative and positive well control. Blank: no serum added; negative: blank mouse serum; positive: previously positive reaction has been identified Mouse serum:);
  • Step 3 Add the enzyme (horseradish peroxidase:)-labeled secondary antibody (anti-antibody) diluted in fresh 1:5000 antibody dilution in the well (all secondary antibodies in ELISA are anti-rat spicy) Root Peroxidase 0.1 ml, incubate at 37 ° C for 60 min, wash 8 times, remove residual liquid, pat dry on paper;
  • Step 4 Add color to the substrate solution: Add 0.1 ml of the temporarily prepared TMB substrate solution to each reaction well, and react at 37 ° C for 10 min in the dark. After the color is complete, add 50 ⁇ l of 2 M sulfuric acid to terminate the reaction;
  • Step 5 Judgment of results: On the ELISA detector, at 450 nm (630 is the reference wavelength:), the 0D value of each well was measured by zeroing the blank control well, and if it was greater than 2.1 times the 0D value of the negative control, it was judged to be positive.
  • the detection results for the ⁇ antigen are shown in Fig. 2.
  • Serum ELISA assays shown in Figure 2 3 weeks after the 2 immunizations of the mice showed that the DNA vaccine plus adjuvant group significantly increased the vaccine-induced Env-specific binding antibody strength compared to the DNA vaccine alone.
  • the results of the test indicate that the HIV Tat adjuvant can significantly increase the intensity of the humoral immune response induced by the DNA vaccine.
  • mice were sacrificed, the mice were immersed in 75% alcohol for disinfection, and operated under sterile ultra-clean bench conditions;
  • Coat Add 50 ⁇ l of coated antibody (purified anti-IFN- ⁇ monoclonal antibody) to each well of ELISPOT plate, supplement the volume to ⁇ with PBS, and cover at 37 °C for 2 h.
  • ELISPOT plate In the ELISPOT plate, add the corresponding antigenic protein of ⁇ (concentration of 10 g/ml), and finally add the lymphocytes obtained above to ⁇ , the total volume to 200 ⁇ 1 (the final concentration of the cells is lx l0 7 / Ml, the total number of cells per well is lx l0 6 ).
  • the negative control was given no peptide and ⁇ RPMI1640 complete medium was added.
  • the positive control was not added with polypeptide, and ⁇ 2.5 ⁇ 1 (25 ⁇ 3 ⁇ 4/ ⁇ 1), myomycin (Inomycin) ⁇ (lg/ml ⁇ immunized group were in duplicate).
  • the adjuvant of Tat adjuvant can significantly enhance the Gag-specific T cell response, and the enhancement ratio is up to 6 times, and there is significant statistical difference.

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  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un adjuvant à base de Tat comprenant (a) un plasmide contenant la séquence d'acide nucléique codant pour la protéine Tat du VIH, un fragment ou une variante de celui-ci ou (b) la protéine Tat du VIH, un fragment ou une variante de celle-ci. L'invention concerne également une composition vaccinale qui comprend un adjuvant à base de Tat, cet adjuvant pouvant augmenter la réponse immunitaire induite par le vaccin.
PCT/CN2011/072665 2011-04-12 2011-04-12 Adjuvant à base de protéine tat du vih et ses utilisations WO2012139281A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005039631A1 (fr) * 2003-10-10 2005-05-06 Istituto Superiore Di Sanita Vaccins contenant la proteine tat du vih en tant qu'adjuvant destine a ameliorer la reponse de lymphocytes t cytotoxiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005039631A1 (fr) * 2003-10-10 2005-05-06 Istituto Superiore Di Sanita Vaccins contenant la proteine tat du vih en tant qu'adjuvant destine a ameliorer la reponse de lymphocytes t cytotoxiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PANG, QIANG ET AL.: "Development of basic study on HIV-1 Tat vaccine.", CHIN J CELLMOL IMMUNOL., vol. 26, no. IS. 2, 2010, pages 192 - 194 *
RICCARDO GAVIOLI ET AL.: "The Tat protein broadens T cell responses directed to the HIV-1 antigens Gag and Env: Implications for the design of new vaccination strategies against AIDS.", VACCINE., vol. 26, no. IS.5, January 2008 (2008-01-01), pages 727 - 737 *

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