WO2011032489A1 - Method of using poxvirus vector hiv vaccine combined with adenoviral vector hiv vaccine and the uses thereof - Google Patents

Abstract

The present invention provides a method of immunizing subjects with poxvirus vector HIV vaccine combined with adenoviral vector HIV vaccine, which method can be used to prevent and treat AIDS as well as other communicable diseases and tumors.

Description

 Combined use of poxvirus vector HIV vaccine and adenovirus vector HIV vaccine method and application thereof

 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.

Background technique

 Acquired Immune Deficiency Syndrome (AIDS) has become one of the most significant challenges to global public health, seriously jeopardizing social progress and economic growth. The World Health Organization (WHO) and 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. In 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. As of the end of October 2007, there were more than 224,000 HIV-infected and AIDS patients reported nationwide. The national epidemiological survey estimated that the total number of HIV-infected people in the country was about 700,000, of which 2007 About 50,000 people are infected. Therefore, the research on AIDS drugs and vaccines will be the top priority of the 11th Five-Year Plan of the Ministry of Science and Technology of China.

 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 phase III clinical trials of two AIDS vaccines in Europe, the United States and Thailand in 2003, the clinical validation of the results of the first phase of development of the AIDS vaccine in the actual sense, showed that the vaccine induced by the antibody alone did not have a real protective effect.

 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. When the virus is infected, 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-γ cytokines alone. (73%), some also secrete TNF-α, but very few cells that secrete IL-2 cytokines or lymphocytes that secrete multiple cytokines at the same time. In addition, the most recent data indicate that a stronger and broader cellular immune response is induced by immunization of Indian rhesus monkeys with different serotypes of adenoviral vectors (rAd26/rAd5) compared to immunization of two Indian rhesus monkeys with Ad5 vector alone. Moreover, more versatile CD8+ and CD4+ T lymphocytes secreting multiple cytokines (IFN-y/TNF-a/IL-2) were also produced. More significantly, after infection of the SIVmac251 virus by intravenous injection, the rAd26/rAd5 combination immunization group controlled the replication and infection of the virus more effectively than the im5 vector alone. 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.

 In summary, combined with recent research and the failure of the Merck vaccine, researchers in this field generally believe that the development of 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. In order to find the ultimate effective HIV vaccine, researchers should experiment with a variety of different types of vaccines in combination to find the best combination.

 Adenoviral 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%).

 As a vaccine against smallpox, the Tiantan strain poxvirus (VTT) has been used in a large number of people in China for a long time. It has excellent safety and is an ideal vaccine carrier. 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. Moreover, experiments have shown that this vector can induce systemic mucosal reactions through mucosal pathways such as nasal and oral.

 Currently, there have been no reports of the use of a recombinant adenoviral vector HIV/SIV vaccine in combination with a modified poxvirus Tiantan strain (MVTT) vector HIV/SIV vaccine.

Summary of the invention

 In order to develop a novel safe and effective HIV vaccine for the prevention and treatment of AIDS, 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. Preferably, in the method, the poxvirus vector HIV vaccine contains the HIV gag, pol, env gene. Preferably, the poxvirus vector HIV vaccine further comprises a helper regulatory protein gene comprising the nef, vpx, vpr, vif, rev and tat genes.

 Preferably, in the method, the adenovirus vector HIV vaccine contains the HIVgag, pol, env gene.

 Preferably, the adenoviral vector HIV vaccine further comprises a helper regulatory protein gene comprising the nef, vpx, vpr, vif, rev and tat genes.

 Preferably, 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.

 Preferably, 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.

 Preferably, 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. .

 In combination with an HIV vaccine using a poxvirus vector or an adenoviral vector alone, the combination of 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 data have shown that strong, broad-spectrum and versatile T lymphocyte responses, especially the killing T lymphocyte response, play a very important role in controlling various viral infections and tumors, and thus in the present invention 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. In addition, since 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 inventor's experimental data laid a solid foundation for this vaccine strategy to enter clinical trials. 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.

 The invention will now be further described with reference to the drawings and embodiments. DRAWINGS

 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;

 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. Test data for secretion of various cytokines by CD8 T cells; 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.

detailed description

 In order to make the present invention easier to understand, the present invention will be further described below in conjunction with the specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention, and the specific experimental methods not mentioned in the following examples are generally carried out according to conventional experimental methods.

 Example 1: Construction of adenovirus and poxvirus vaccine carrying SIVgag, pol and env genes and immunogenicity in mice

 1. Acquisition of SIVmac239 gene

 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.

 1.1 The optimized sequence of the SIVmac239gag gene used in this example is as follows:

Cgaagcttac catgggcgtg aggaactctg tgctgtctgg caagaaggct gatgagctgg 60

Agaagatcag gctgaggccc aatggcaaga agaagtacat gctgaagcat gtggtgtggg 120

Ctgccaatga gctggacagg tttggcctgg ctgagtccct gctggagaac aaggagggct 180

Gccagaagat cctgtctgtg ctggcccccc tggtgcccac aggctctgag aacctgaagt 240

Ccctgtacaa cacagtgtgt gtgatctggt gcatccatgc tgaggagaag gtgaagcaca 300

Cagaggaggc caagcagatt gtgcagaggc acctggtggt ggagacaggc accacagaga 360

Ccatgcccaa gacctccagg cccacagccc cctcctctgg cagggggggc aactaccctg 420

Tgcagcagat tgggggcaac tatgtgcacc tgcccctgtc ccccaggacc ctgaatgcct 480

Gggtgaagct gattgaggag aagaagtttg gggctgaggt ggtgcctggc ttccaggccc 540

Tgtctgaggg ctgcaccccc tatgacatca accagatgct gaactgtgtg ggggaccacc 600

Aggctgctat gcagatcatc agggacatca tcaatgagga ggctgctgac tgggacctgc 660

Agcaccccca gcctgccccc cagcagggcc agctgaggga gccctctggc tctgacattg 720

Ctggcaccac ctcctctgtg gatgagcaga tccagtggat gtacaggcag cagaacccca 780

Tccctgtggg caacatctac aggaggtgga tccagctggg cctgcagaag tgtgtgagga 840

Tgtacaaccc caccaacatc ctggatgtga agcagggccc caaggagccc ttccagtcct 900 acgtggacag gttctacaag tccctgaggg ctgagcagac agatgctgct gtgaagaact 960 ggatgaccca gaccctgctg atccagaatg ccaaccctga ctgcaagctg gtgctgaagg 1020 gcctgggggt gaaccccacc ctggaggaga tgctgacagc ctgccagggg gtggggggcc 1080 ctggccagaa ggccaggctg atggctgagg ccctgaagga ggccctggcc cctgtgccca 1140 tcccctttgc tgctgcccag cagaggggcc ccaggaagcc catcaagtgc tggaactgtg 1200 gcaaggaggg ccactctgcc aggcagtgca gggcccccag gaggcagggc tgctggaagt 1260 gtggcaagat ggaccatgtg atggccaagt gccctgacag gcaggctggc ttcctgggcc 1320 tgggcccctg Gggcaagaag cccaggaact tccccatggc ccaggtgcac cagggcctga 1380 tgcccacagc cccccctgag gaccctgctg tggacctgct gaagaactac atgcagctgg 1440 gcaagcagca gagggagaag cagagggagt ccagggagaa gccctacaag gaggtgacag 1500 aggacctgct gcacctgaac tccctgtttg ggggggacca gtaaagtaaa gcccgggtct 1560 agagg 1565

 1.2 The optimized sequence of the SIVmac239pol gene used in this example is as follows:

gatccaccat ggtgctggag ctgtgggaga ggggcaccct gtgcaaggcc atgcagtccc 60 ccaagaagac aggcatgctg gagatgtgga agaatggccc atgctatggc cagatgccca 120 ggcagacagg cggcttcttc aggccatggt ccatgggcaa ggaggccccc cagttccccc 180 atggctcctc tgcctctggc gctgatgcca actgctcccc caggggccca tcctgtggct 240 ctgccaagga gctgcatgct gtgggccagg ctgctgagag gaaggctgag aggaagcaga 300 gggaggccct gcagggcggc gacaggggct ttgctgcccc ccagttctcc ctgtggagga 360 ggcctgtggt gacagcccac attgagggcc agcctgtgga ggtgctgctg gacacaggcg 420 ctgatgactc cattgtgaca ggcattgagc tgggccccca ctacaccccc aagattgtgg 480 gcggcattgg cggcttcatc aacaccaagg agtacaagaa tgtggagatt gaggtgctgg 540 gcaagaggat caagggcacc atcatgacag gcgacacccc catcaacatc tttggcagga 600 acctgctgac agccctgggc atgtccctga acttccccat tgccaaggtg gagcctgtga 660 aggtggccct gaagcctggc aaggatggcc ccaagctgaa gcagtggccc ctgtccaagg 720 agaagattgt ggccctgagg gagatctgtg agaagatgga gaaggatggc cagctggagg 780 aggccccccc caccaaccca tacaacaccc ccacctttgc catcaagaag aaggacaaga 840 acaagtggag gatgctgatt gacttcaggg agctgaacag ggtgacccag gacttcacag 900 aggtgcagct gggcatcccc catcctgctg gcctggccaa gaggaagagg atcacagtgc 960 tggacattgg cgatgcctac ttctccatcc ccctggatga ggagttcagg cagtacacag 1020 ccttcaccct gccatctgtg aacaatgctg agcctggcaa gaggtacatc tacaaggtgc 1080 tgccccaggg ctggaagggc tcccctgcca tcttccagta caccatgagg catgtgctgg 1140 agccattcag gaaggccaac cctgatgtga ccctggtgca gtacatggat gacatcctga 1200 ttgcctctga caggacagac ctggagcatg acagggtggt gctgcagtcc aaggagctgc 1260 tgaactccat tggcttctcc acccctgagg agaagttcca gaaggacccc ccattccagt 1320 ggatgggcta tgagctgtgg cccaccaagt ggaagctgca gaagattgag ctgccccaga 1380 gggagacctg gacagtgaat gacatccaga agctggtggg cgtgctgaac tgggctgccc 1440 agatctaccc tggcatcaag accaagcatc tgtgcaggct gatcaggggc aagatgaccc 1500 tgacagagga ggtgcagtgg acagagatgg ctgaggctga gtatgaggag aacaagatca 1560 tcctgtccca agagcaggag ggctgctact accaggaggg caagcccctg gaggccacag 1620 tgatcaagtc ccaggacaac cagtggtcct acaagatcca tcaggaggac aagatcctga 1680 aggtgggcaa gtttgccaag atcaagaaca cccacaccaa tggcgtgagg ctgctggccc 1740 atgtgatcca gaagattggc aaggaggcca ttgtgatctg gggccaggtg cccaagttcc 1800 atctgcctgt ggagaaggat gtctgggagc agtggtggac agactactgg caggtgacct 1860 ggattcctga gtgggacttc atctccaccc cccccctggt gaggctggtc ttcaacctgg 1920 tgaaggaccc cattgagggc gaggagacct actacacaga tggctcctgc aacaagcagt 1980 ccaaggaggg caaggctggc tacatcacag acaggggcaa ggacaaggtg aaggtgctgg 2040 agcagaccac caaccagcag gctgagctgg aggccttcct gatggccctg acagactctg 2100 gccccaaggc caacatcatt gtggactccc agtatgtgat gggcatcatc acaggctgcc 2160 ccacagagtc tgagtccagg ctggtgaacc agatcattga ggagatgatc aagaagtctg 2220 agatctatgt ggcctgggtg cctgcccaca agggcattgg cggcaaccag gagattgacc 2280 atctggtctc ccagggcatc aggcaggtgc tgttcctgga gaagattgag cctgcccagg 2340 aggagcatga caagtaccac tccaatgtga aggagctggt cttcaagttt ggcctgccca 2400 ggattgtggc caggcagatt gtggacacct gtgacaagtg ccatcagaag ggcgaggcca 2460 tccatg gcca ggccaactct gacctgggca cctggcagat ggactgcacc catctggagg 2520 gcaagatcat cattgtggct gtgcatgtgg cctctggctt cattgaggct gaggtgatcc 2580 cccaggagac aggcaggcag acagccctgt tcctgctgaa aggtggccca gctggctggc tgcactgcat gaacttcaag aggaggggcg 2640 tcacccatct gcacacagac aatggcgcca actttgcctc ccaagaggtg aagatggtgg 2700 cctggtgggc tggcattgag cacacctttg gcgtgccata caacccccag tcccagggcg 2760 tggtggaggc catgaaccat catctgaaga accagattga caggatcagg gagcaggcca 2820 actctgtgga gaccattgtg ctgatggctg 2880 gcattggcga catgacccct gctgagaggc tgatcaacat gatcaccaca gagcaggaga 2940 tccagttcca gcagtccaag aactccaagt tcaagaactt cagggtctac tacagggagg 3000 gcagggacca gctgtggaag ggccctggcg agctgctgtg gaagggcgag ggcgctgtga 3060 tcctgaaggt gggcacagac atcaaggtgg tgcccaggag gaaggccaag atcatcaagg 3120 actatggcgg cggcaaggag gtggactcct cctcccacat ggaggacaca ggcgaggcca 3180 gggaggtggc tgactacaag gatgatgatg acaagtaaat ctagagg 3227

1.3 The optimized sequence of the SIVmac239env gene used in this study is as follows:

cgggatccac catgggctgc ctgggcaacc agctgctgat tgccatcctg ctgctgtctg 60 tctatggcat ctactgcacc ctgtatgtga cagtcttcta tggcgtgcct gcctggagga 120 atgccaccat ccccctgttc tgtgccacca agaacaggga cacctggggc accacccagt 180 gcctgcctga caatggcgac tactctgagg tggccctgaa tgtgacagag tcctttgatg 240 cctggaacaa cacagtgaca gagcaggcca ttgaggatgt ctggcagctg tttgagacct 300 ccatcaagcc atgtgtgaag ctgtcccccc tgtgcatcac catgaggtgc aacaagtctg 360 agacagacag gtggggcctg accaagtcca tcaccaccac agcctccacc acctccacca 420 cagcctctgc caaggtggac atggtgaatg agacctcctc ctgcattgcc caggacaact 480 gcacaggcct ggagcaggag cagatgatct cctgcaagtt caacatgaca ggcctgaaga 540 gggacaagaa gaaggagtac aatgagacct ggtactctgc tgacctggtc tgtgagcagg 600 gcaacaacac aggcaatgag tccaggtgct acatgaacca ctgcaacacc tctgtgatcc 660 aggagtcctg tgacaagcac tactgggatg ccatcaggtt caggtactgt gccccccctg 720 Gctatgccct gctgaggtgc aatgacacca actactctgg cttcatgccc aagtgctcca 780

Aggtggtggt ctcctcctgc accaggatga tggagaccca gacctccacc tggtttggct 840

Tcaatggcac cagggctgag aacaggacct acatctactg gcatggcagg gacaacagga 900

Ccatcatctc cctgaacaag tactacaacc tgaccatgaa gtgcaggagg cctggcaaca 960

Agacagtgct gcctgtgacc atcatgtctg gcctggtctt ccactcccag cccatcaatg 1020

Acaggcccaa gcaggcctgg tgctggtttg gcggcaagtg gaaggatgcc atcaaggagg 1080

Tgaagcagac cattgtgaag catcccaggt acacaggcac CBBC3BC3CB gacaagatca 1140

Acctgacagc ccctggcggc ggcgaccctg aggtgacctt catgtggacc aactgcaggg 1200

Gcgagttcct gtactgcaag atgaactggt tcctgaactg ggtggaggac aggaacacag 1260

Ccaaccagaa gcccaaggag cagcacaaga ggaactatgt gccatgccac atcaggcaga 1320

Tcatcaacac ctggcacaag gtgggcaaga atgtctacct gccccccagg gagggcgacc 1380

Tgacctgcaa ctccacagtg acctccctga ttgccaacat tgactggatt gatggcaacc 1440

Agaccacat caccatgtct gctgaggtgg ctgagctgta caggctggag ctgggcgact 1500

Acaagctggt ggagatcacc cccattggcc tggcccccac agatgtgaag aggtacacca 1560

Caggcggcac ctccaggaac aagaggggcg tctttgtgct gggcttcctg ggcttcctgg 1620

Ccacagctgg ctctgccatg ggcgctgcct ccctgaccct gacagcccag tccaggaccc 1680

Tgctggctgg cattgtgcag cagcagcagc agctgctgga tgtggtgaag aggcagcagg 1740

Agctgctgag gctgacagtc tggggcacca agaacctgca gaccagggtg acagccattg 1800

Agaagtacct gaaggaccag gcccagctga atgcctgggg ctgtgccttc aggcaggtct 1860

Gccacaccac agtgccatgg cccaatgcct ccctgacccc caagtggaac aatgagacct 1920

Ggcaggagtg ggagaggaag gtggacttcc tggaggagaa catcacagcc ctgctggagg 1980

Aggcccagat ccagcaggag aagaacatgt atgagctgca gaagctgaac tcctgggatg 2040

Tctttggcaa ctggtttgac ctggcctcct ggatcaagta catccagtat ggcgtctaca 2100

Ttgtggtggg cgtgatcctg ctgaggattg tgatctacat tgtgcagatg ctggccaagc 2160

Tgaggcaggg ctacaggcct gtcttctcct cccccccatc ctacttccag cagacccaca 2220

Tccagcagga ccctgccctg cccaccaggg agggcaagga gagggatggc ggcgagggcg 2280

Gcggcaactc ctcctggcca tggcagattg agtacatcca cttcctgatc aggcagctga 2340

Tcaggctgct gacctggctg ttctccaact gccggaccct gctgtccagg gtctaccaga 2400

Tcctgcagcc catcctgcag aggctgtctg ccaccctgca gaggatcagg gaggtgctga 2460

Ggacagagct gacctacctg cagtatggct ggtcctactt ccatgaggct gtgcaggctg 2520

Tctggaggtc tgccacagag accctggctg gcgcctgggg cgacctgtgg gagaccctga 2580

Ggaggggcgg caggtggatt ctggccatcc ccaggaggat caggcagggc ctggagctga 2640

Ccctgctgga ctacaaggat gatgatgaca agtaaatcta gage 2684

 2. Construction, amplification, purification and identification of recombinant adenovirus

According to the conventional recombinant adenovirus construction method, 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. After amplification, 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 results of the detection of white expression levels; as shown in Figure 1, 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. For example, Ad5-gag can express P55 and P27, Ad5-pol can express P66/51, P31, P10, etc. Ad5-env can express gpl 60, gpl20, gp41.

 3. Carrying adenoviral vector Immunogenicity of SIV full antigen group vaccine in mice

Further, the immunogenicity of these vaccines was verified in mice with 10 6-8 female C57BL/6 mice per group. The immunization dose was lx l0 ^1G vp recombinant adenovirus/only/ΙΟΟμΙ^, and each of the four sides of the mice was injected with 50 μί. A total of 2 immunizations, 2 weeks apart. Figure 2 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. 2Α and 2Β, the spleens of the mice were taken out after primary immunization and booster immunization, and mixed together in groups to prepare spleen cells for ELISPOT detection. A strong IFN-y ELISPOT immune response was detected in each of the immunized groups, and the response level to SIV Gag and Pol was relatively higher. More significantly, in the fifth group, that is, 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. Although the response to a single antigen has decreased, we can also see that the overall response level for each antigen is more consistent, that is, the response to each antigen is more coordinated, avoiding excessive responses to some antigens, which may More conducive to control the replication of SIV virus. An immune response against any SIV antigen was not detected in the control mice.

 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

1. Experimental materials

 1.1 experimental animals

 16 Chinese rhesus monkeys were purchased from the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences and the South China Endangered Animal Research Institute to establish a non-human primate disease model research base with a body weight of 3-5kg and an age of 3 -6 years. Both male and female. These monkeys have been tested before the experiment. Nitrate tuberculosis, Shigella, Salmonella, STLV-1, SIV, SRV, etc. are all negative. The operation of the animal experiment is in accordance with the relevant regulations for the use of experimental animals.

 1.2 virus

 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).

 1.3 flow antibody

 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.

 1.4 SIV peptides of various antigens

All 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.

 1.5 other reagents

 ELISPOT board (brand: Millipore; article number: MSIPS4510) was purchased from Daktronics Biotech Co., Ltd., SIV virus particle lysate was prepared for itself, and TMB/E substrate (Cat. No. ES001) was purchased from Chemicon, USA. BCIP/NBT substrate (brand: Pierce; article number: 34042) was purchased from Guangzhou Jitai Biotechnology Co., Ltd. Cell dye CFSE was purchased from Molecule Probe. Streptomycin-conjugated alkaline phosphatase (brand: BD PharMingen, Cat. No. 554065) was purchased from Gene Co., Ltd.

 2, the real insurance method

 2.1 Animal grouping and immunization strategies

Sixteen monkeys were divided into 4 groups, 4 monkeys in each group. As shown in Figure 3, the combination of poxvirus vector (MVTT) SIV vaccine and adenovirus (Ad5) vector SIV vaccine in rhesus monkeys was used for immunization and challenge. . 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.

 2.2 Multi-color flow technology for detecting multifunctional T cells

1) 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.

 2) Add specific antigen peptide to the above cell suspension, add the corresponding concentration of DMSO to the negative control well, and add phorbol ester (PMA) (20 ng/ml) + ionomycin (1000 ng/ml) to the positive control.

3) Incubate at 37 °C, 5% CO _{2 for} 1-2 h.

 4) Add brefeldin (BFA) ( ΙΟμ^ηιΙ) and mix.

5) Incubate at 37 °C, 5% CO _{2 for} 16 h.

 6) Blow even cells and transfer to a flow tube (12x75mm polystyrene tube). Add 3 ml of flow wash. After mixing, the mixture was centrifuged at 300 g for 10 minutes at room temperature.

 7) Discard the supernatant. The addition of cells separately indicated that the labeled antibodies, such as CD3 - pacific blue, CD4-AmCyan, CD8-APC-Cy7, CD28-FITC, CD95-PE-Cy5, were shaken for 2-3 seconds.

 8) Leave at room temperature for 25 _ 30 minutes.

 9) Add 3ml FACS lotion. After mixing, centrifuge at room temperature for 300 minutes at room temperature for 10 minutes.

 10) Repeat the washing once.

 11) Shake the cells evenly, then add 250μ1 cell fix / permeate (cytofix/cytoperm, brand: BD; Cat. No.: 554722), mix.

 12) 4 ° C, protected from light for 20 min.

 13) Add 1 ml of 1 χ cell permeabilization/washing solution (perm/wash, brand: BD Biosciences; article number: 554723. The product is 10 times, diluted to 1 time with sterile distilled water), 400 g, and centrifuged for 8 min. Discard the supernatant.

 14) Repeat once.

 15) Discard the supernatant, add IFNy-PE/TNFa-PE CY7/IL2-APC (10 ul each), and mix.

 16) 4° (protected from light for 30_60 minutes.

17) Add lml lx fine pack perme/wash (perm/wash), 400g, and centrifuge for 8min. 18) Discard the supernatant and add 3 ml of flow wash. After mixing, centrifuge at room temperature for 10 minutes at 400 g.

 19) Aspirate most of the supernatant, leaving approximately 300μ1. Shake and mix and put on the machine.

 20) Detection on a FACSAria flow cytometer, data acquisition using Cell Quest software, and analysis using Modfit software.

 2.3 IFN-γ enzyme-linked immunospot technique

 first day:

 Take a tube of coated antibody (anti-monkey IFN-γ antibody containing this antibody, brand: U-Cytech, clone number MD-1) diluted with sterile PBS (pH 7.2-7.4) 1: 100, ΙΟΟμΙ / Wells were added to 96-well PVDF membrane plates and coated overnight at 4 °C.

 the next day:

 1 Remove the liquid, wash it 3 times with sterile PBS, add 200 μl of R10 complete medium to each well, and block at 37 °C for 2 hours.

 2 PBMC were separated during the blocking period.淋巴 Separate lymphatics with a 95% diluted lymphocyte separation (trade name opti-prep).

 3 Discard the blocking solution and add 100μ1 每 to each well.

 4 In the experiment, a positive control group (ConA group), a specific peptide, and a blank control (DMSO) were set. Incubate in a 37 ° C 5% CO 2 incubator.

 Third day:

 After 24 h, the cell suspension was discarded and washed 6 times with sterile PBST 220μ1/Ανε11.

 6 Remove the lotion and dry it on sterile dry paper.

 7 The test antibody (biotinylated anti-monkey IFN-γ antibody) was diluted 1:400 with PBST containing 5 % FBS, ΙΟΟμΙ/well, overnight at 4 °C.

 Fourth day:

 1 Wash 4 times with sterile PBST 220μ1/well.

 2 Remove the lotion and dry it on sterile dry paper.

 3 Configure streptomycin-conjugated alkaline phosphatase: Streptomycin-conjugated alkaline phosphatase was diluted 1:2500 with PBST containing 5 % FBS, ΙΟΟμΙ/well, 37 ° C, for 2 hours.

 4 Dispose of BCIP/NBT substrate (Pierce, Cat: 34042), warm bath at 37 °C for 30 min.

 5 Wash 5 times with sterile PBST 220 l/well, remove the wash, and dry on sterile dry paper.

 6 Add BCIP/NBT substrate, ΙΟΟμΙ/well, and avoid light reaction for 7 min.

 7 Discard the substrate and wash twice with water. Air dry reading board.

 2.4 CFSE detection of lymphocyte proliferation

 1) Isolation of PBMC cells from rhesus monkeys.

2) Centrifuge for 10 minutes at 300 g after cell counting. The supernatant was discarded, was added 0.1% FBS / PBS liquid resuspended, washed once, then the number of the appropriate volume of 0.1% FBS / PBS and resuspended to give 4xl0 ⁶ / ml of cell suspension.

 3) Dilute the CFSE stock solution to 2 mol/L with 0.1% FBS/PBS. The following operations are as far as possible from light.

4) Take 0.25 ml of the above cell suspension and CFSE dilution, and mix gently. (At this time, the cells are fragile, and the movement should be as gentle as possible!)

 5) Leave at 37 °C for 10 min.

6) Add ice-cold RPMI 1640 complete medium and freeze for 5 minutes to stop staining. 7) Centrifuge at 300g for 5 minutes at room temperature. The supernatant was discarded and washed twice with water-cooled complete 1640 medium containing 10% FBS. Finally, it was resuspended in 1 ml of R10 medium.

8) The above cell suspension was added to a 24-well culture plate at 1 ml per well, and stimulated with SEB (final concentration lug/ml) and a specific polypeptide library (2 _g / ml), respectively.

 9) Culture at 37 °C, 5 % C02 in the dark for 5-6 days.

 10) Blow even cells and transfer to a flow tube (12x75mm polystyrene tube). Add 4 ml of FACS lotion. After mixing, centrifuge at room temperature for 10 minutes at 250 g.

 11) The vacuum pump pumps off the supernatant. Oscillating on a vortex shaker for 2 - 3 seconds.

 12) Add 20ul CD4-PE, CD8-Percp, CD3-APC respectively. Oscillate for 2 - 3 seconds.

 13) Leave at room temperature for 25 - 30 minutes.

 14) Add 4ml FACS lotion. After mixing, centrifuge at room temperature for 300 minutes at room temperature for 10 minutes.

 15) Aspirate most of the supernatant, leaving about 300ul. Shake well. Directly on the machine. Or continue with step 15.

 16) Add 30ul of 10% formalin (Brand: Polysciences; Cat. No. 04018) and shake for 10 - 20 seconds.

 17) 4 Keep away from light and check on the machine within 24 hours.

 18) Detection on a FACScalibur flow cytometer, data acquisition using Cell Quest software, and analysis using Modfit software.

 2.5 Data Analysis

 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.

 3, the experimental results

 3.1 MVTT /Ad5 co-immunization strategy induces a stronger and more durable cellular immune response in Chinese rhesus monkeys. We compared the immunogenicity of monkeys alone or in combination with MVTT vector and Ad5 vector. The results showed that Ad5 was immunized once. The vector-induced immune response is much higher than the MVTT vector, and the combined use of these two vectors is far more immune than using either vector alone. Figure 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. As shown in Figure 4, we observed that a modified vaccinia virus Tiantan strain (MVTT) vector was immunized alone, and a certain degree of cellular immune response was observed in monkeys, and an average of 237 stimuli were generated for the antigens carried by the target. Spots (million PBMC cells); Once we immunized once, we constructed a type 5 adenoviral vector, which produced a strong cellular immune response in monkeys, and generated an average of 2643 spots for the stimulation of the antigen of interest. Ten thousand PBMC cells). When priming with the MVTT vector and boosting with the Ad5 vector after 6 weeks, a high level of cellular immune response was induced in the monkey, and the stimulation of the antigen of interest carried by the average produced 7383 spots (million PBMC). cell). Therefore, this strategy is on average 31 times more effective than the response to cell immunity induced by the MVTT vector alone, and 2.8 times more than the response level of the Ad5 vector alone. This strategy not only induces a very high level of cellular immune response in monkeys, but more importantly, this response lasts for a long time. After 21 weeks from the last immunization, monkeys in the co-immunization group were still able to detect a high level of ELISPOT response.

3.2 MVTT /Ad5 combined immunization strategy induced more versatile memory lymphocytes in Chinese rhesus monkeys Although IFN-γ 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-γ, including IL-2, TNFa, MIP 1 β 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. The results are shown in Fig. 5. 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. Specifically, in the monkeys immunized with MVTT and Ad5 vector, 0.2464±0.2770% of CD8+ T lymphocytes secrete three cytokines of IFN-γ/TNF-a/IL-2, 1.4668±1.1845% of CD8+ T lymphocytes secrete both IFN-γ/TNF-a cytokines; while only 0.0320 ± 0.0472% of CD8+ T lymphocytes secrete IFN-γ/TNF-a /IL-2 in monkeys immunized with Ad5 vector alone. These three cytokines, 0.1587 ± 0.2035% of CD8+ T lymphocytes secrete two cytokines, IFN- y / TNF-a. That is, compared with Ad5 vector alone, MVTT combined with Ad5 vector produced 7.7 times more multifunctional CD8+ T lymphocytes that secrete IFN-γ/TNF-a/IL-2 simultaneously. More than 9 times more multifunctional CD8+ T lymphocytes capable of secreting both cytokines of IFN-γ/TNF-a.

 We further examined the ability of specific memory cells to produce and secrete cytokines after combined immunization with MVTT and Ad5 vectors. The results are shown in Fig. 6. 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. The ability of SIV antigen-specific memory CD4+ T cells and CD8+ T cells to produce and secrete cytokines is significantly stronger in these monkeys. Compared with the other groups, 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.

 3.3 MVTT/Ad5 combined immunization strategy Inducing stronger lymphocyte proliferation ability in Chinese rhesus monkeys, it shows that CD4+ T cells and CD8+ T cells in patients with long-term non-progression of HIV have strong in vitro proliferation against HIV antigen. The ability of CD4+ T cells and CD8+ T cells in HIV-infected patients to target HIV antigens is weak in vitro. Moreover, this proliferative capacity has a negative correlation with the virus copy number in the patient's plasma, and is positively correlated with the number of CD4+ T cells. Therefore, the ability to detect the proliferation of T lymphocytes can better reflect the degree of control of HIV replication in the body.

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. Therefore, compared with the Ad5 vector alone, the proliferation ability of CD4+ T lymphocytes against antigen-stimulated cells was slightly enhanced after MVTT was combined with the Ad5 vector. 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. Thus, compared to the Ad5 vector alone, the proliferation of CD8+ T lymphocytes against antigen-stimulated MVTT was significantly increased by 2.5-fold compared with the Ad5 vector.

 In summary, 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

1 experimental materials:

 1.1 Experimental animals Same as Example 2.

 1.2 virus

 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.

 1.3 antibody

 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.

 1.4 other reagents

 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.

 2 experimental methods

 2.1 Animal grouping and challenge experiments

Sixteen monkeys were divided into 4 groups, 4 monkeys in each group. 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.

 2.2 Quantitative PCR determination of SIV viral load

 1) Plasma is separated by conventional methods.

 2) Extraction of SIV virus RNA in plasma.

 3) Perform a one-step fluorescence quantification RT-CR, and perform a standard curve for each test.

 4) Configure the reaction system (using QuantiTect's SYBR Green RT-PC kit, article number: 204243):

 2x QuantiTect SYBR Green RT-PCR main reaction solution 25 μΐ

QuantiTect RT mix 0.5μ1 The kit comes with primer 1 (ΙΟμΜ) 0.5μ1

 The kit comes with primer 2 (ΙΟμΜ) 0.5μ1

 RNA template 2μ1

 RNase-free water 21·5μ1

 Total volume 50μ1

 5) Arrange the test layout according to the number of samples. The RT-PCR reaction conditions were then carried out according to the following conditions: a) 50 ° C for 30 min

 b) 95 ° C 15 min

 c) 94 °C 15s

 d) 60 °C 30s

 e) 72 °C 30s

 f) reading the board;

 g) 3)-6) cycle for 45 weeks;

 h) 65-95 degrees to make the melting curve, read every 0.5 °C for 1 second;

 i) kept at 24 ° C;

 6) According to the standard curve, 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.

 2.3 SIV virus particle lysate ELISA method for detection of binding antibodies

 I) Coating antigen: 96μΙ antigen (1 g/ml, diluted with PBS pH 7.4 or carbonate buffer of pH 9.6) was added to each well of a 96-well plate and coated overnight at 4 °C.

 2) Washing the plate: The antigen was discarded the next day, dried, and washed 3 times with PBS.

 3) Blocking: PBST containing 5% non-fat dry milk powder, 250 μl per well, blocked at 37 ° C for 1 h.

 4) Wash the plate: Add 20 (^1 PBST, 3 min/time, 3 times per well, moisturize and store for use.

 5) Add the specimen to be tested: Add ΙΟΟμΙ to each well, and make a double-well assay for each sample, and react at 37 °C for 2 h. (If dilution is required, use a blocking solution)

 6) Wash the plate: Add 200μ1 ΡΒ8Τ per well, 3min/time, and wash 6 times.

 7) Binding of the second antibody: 每μΙ appropriate dilution of HRP-anti-mouse IgG (1: 8000) antibody or anti-human IgG (l:4000) was added to each well and incubated at 37 °C for 1 h. (diluted with blocking solution)

 8) Wash the plate: Add 200μ1 ΡΒ8Τ per well, 3min/time, and wash 6 times.

 9) Color development: Add ΙΟΟμΙ enzyme reaction substrate to each well, and let it stand for 5-10 minutes at room temperature.

10) The reaction was stopped by the addition of an equal volume of 1 M H ₂ SO ₄ .

II) Determination of results: Determination of OD ₄₅₀ value at 450 nm by ELISA plate spectrophotometer

 2.4 See Example 2 for other methods.

 3, the experimental results

 3.1 The cellular immune response to each antigen after rhesus monkey challenge

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. As shown in Fig. 8A, after the SIVmac239 virus challenge, 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. More interestingly, 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. We speculate that this is due to the SIV virus in these monkeys. 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.

 3.2 Antibody response to SIV after rhesus monkey challenge

 We also tested antibodies in serum from these experimental monkeys at different times. As shown in Figure 9, the SIV-specific antibody response levels before and after SIVmac239 infection in monkeys were detected by ELISA, whether MVTT vector or Ad5 vector, after primary immunization. Most monkeys have low antibody levels, but most monkeys have elevated antibody levels after booster immunization. More significantly, antibody levels in the immunized group rapidly rose to very high levels after being challenged by the virus. In the control group, the test monkeys did not detect antibodies before the challenge, and the antibody production time was relatively slow after the challenge, and the titer was low.

 3.3 Determination of viral load in rhesus monkeys

 After challenge, the viral load of all monkeys peaked in 10-14 days. As can be seen from Figure 10, the peak viral load in the monkeys of the combined immunization group was significantly lower than that of the control group. In the combined immunization group, 1 of the 4 monkeys had not detected the SIV virus until now, and the other 3 viral loads were reduced by 1.371 og compared with the control group. This demonstrates that our combination strategy effectively controls SIV infection and replication during acute infection, and the long-term immunoprotective effect of this method requires longer observation times to confirm. One thing that needs special mention is that we use high-dose mucosal infections of the more difficult-to-neutral SIVmac239 strain, while most of the previous studies used relatively virulent strains. It should be noted that the above embodiments are only intended to illustrate the technical solutions of the present invention, and are not intended to limit the scope of the present invention. The technical solutions of the present invention may be modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims

Claim

1. Combination of poxvirus vector HIV vaccine and adenovirus vector The method of HIV vaccine is characterized in that a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine is used to immunize the body.

The method of using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to claim 1, wherein the body is first immunized with a poxvirus vector HIV vaccine, and then the adenovirus vector HIV vaccine is used to immunize the body.

The method of using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to claim 1, wherein the body is first immunized with an adenovirus vector HIV vaccine, and the body is immunized with a poxvirus vector HIV vaccine.

The method according to claim 1, wherein the poxvirus vector HIV vaccine comprises HIV gag, pol and env genes.

The method of using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to claim 4, wherein the poxvirus vector HIV vaccine further comprises a helper regulatory protein gene, the helper regulatory protein Genes include the nef, vpx, vpr, vif, rev and tat genes.

6. The combination of a poxvirus vector HIV vaccine and an adenovirus vector according to claim 1 A method of vaccine, characterized in that the adenoviral vector HIV vaccine contains HIV gag, pol and env genes.

The method of using a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to claim 6, wherein the adenovirus vector HIV vaccine further comprises a helper regulatory protein gene, the helper regulatory protein Genes include the nef, vpx, vpr, vif, rev and tat genes.

The method according to claim 1, wherein the pathway of the immune body comprises a mucosal route, an intramuscular route, and an intravenous route.

The method according to claim 1, wherein the adenoviral vector is a type 5 adenovirus vector.

The method according to claim 1, wherein the poxvirus vector is a modified Tiantan strain poxvirus vector.

11. Use of a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to any one of claims 1 to 10 for the prevention and treatment of AIDS.

The use of a combination of a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine according to any one of claims 1 to 10 for the prevention and control of hepatitis B, Ebola virus and tumors.