CN101670102A - Method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine and application - Google Patents

Abstract

The invention provides a method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine, and in particular relates to a method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine to immunize organisms; the method is that: the poxvirus vector HIV vaccine is used for immunizing first and then the adenoviral vector HIV vaccine is used for immunize organisms; or the adenoviral vector HIV vaccine is used first for immunizing organism first and then the poxvirus vector HIV vaccine is used for immunizing organism. The invention also provides the applicationof the method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine on the preventive and therapeutic HIV vaccines field, so as to prevent and treat AIDS and Hepatitis B, Ebolavirus and tumor. The method can induce strong, broad-spectrum and multi-functional T leukomonocyte reaction and can be widely applied to preventing and treating AIDS and other communicable diseases and tumor.

Description

Method and application of combined use of poxvirus vector HIV vaccine and adenovirus vector HIV vaccine

technical field

The invention relates to the field of biotechnology, in particular to a new method for preventing and treating AIDS by using a poxvirus vector HIV vaccine and an adenovirus vector HIV vaccine in combination and its application.

Background technique

AIDS (Acquired Immune Deficiency Syndrome, AIDS) has become one of the most significant challenges to global public health, seriously endangering social progress and economic growth. The World Health Organization (WHO) and the United Nations AIDS Program (UNAIDS) announced at the end of 2008 that the total number of HIV-infected people in the world is about 33 million, and the cumulative 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 people were infected with HIV; Asia and Eastern Europe have become the regions where HIV spreads most rapidly (UNAIDS report, 2008). The trend of HIV prevalence in my country is also quite serious. As of the end of October 2007, more than 224,000 cases of HIV-infected and AIDS patients have been reported nationwide. The national epidemiological survey estimates that the total number of surviving HIV-infected people in the country is about 700,000. About 50,000 people were infected. Therefore, research on AIDS prevention and treatment drugs and vaccines will be the top priority of the "Eleventh Five-Year Plan" of the Ministry of Science and Technology of my country.

Although many advances have been made in drug treatment and control of AIDS in the past one or two decades, 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 appeared. , protease inhibitors, etc.) and new therapy (combined cocktail method), but once the body is infected, the HIV virus cannot be eradicated, and due to the high variability, the drug resistance of the HIV virus is becoming more and more serious, and drug treatment brings HIV infection. The prospect of these therapies is unsatisfactory due to the large adverse reactions and the economic affordability of patients. Therefore, the development of effective HIV vaccines is still the most urgent need. In the field of HIV vaccines, it is generally believed that HIV vaccine research can be divided into three stages:

The first stage: The AIDS vaccine research that started in 1984 focused on inducing antibodies to prevent viral infection as the main goal, without considering the role of cellular immunity. Two phase III clinical trials of AIDS vaccines completed in Europe, America and Thailand in 2003, that is, the clinical verification of the research results of the first stage of AIDS vaccine development in the actual sense, showed that vaccines that simply induce antibody production do not have real protective effects.

The second stage: AIDS vaccine research emphasizes the role of cellular immune response. At this stage, recombinant virus vector vaccines (vaccinia, adenovirus, canarypox virus, etc.) that induce cellular immune responses are the mainstays, because theoretically, cellular immune responses can effectively control HIV virus replication and infection, and mathematical models also show that, Reducing the viral load by 1 log value can effectively reduce the transmission rate of the population, which allows clinical trials to see the application prospects of this vaccine. One of the most notable achievements is the AIDS vaccine developed by Merck, which uses human adenovirus type 5 as the carrier. In the SHIV89.6P model, it has been proved that the induction of CD8 T cell immune response against HIV has a better effect. Protective effects. Subsequently, Merck Company pushed the vaccine into clinical trials, and entered clinical phase IIb in 2004. However, according to the clinical trial results published in September 2007, the simple emphasis on cellular immune response cannot produce protective effects.

The currently more popular view, the third stage view, holds that an effective AIDS vaccine needs to induce a balanced humoral and cellular immune response at the same time. Antibodies, as the first line of defense during virus infection, can neutralize part of the virus, which buys time for the subsequent cell-mediated memory response to be activated, while a strong cellular immune response can clear virus-infected cells and reduce the viral load, thereby It can reduce the transmission rate of HIV virus in the population.

McElrath et al systematically analyzed Merck's HIV clinical trial data, and found that although the vaccine did not play a protective role, the vaccine produced a strong immune response in most of the tested population (77%), and the analysis also found that these The specific CD8+ T cells in the population mainly secrete IFN-γ cytokines alone (73%), and some secrete TNF-a, but very few cells secrete IL-2 cytokines or secrete multiple cytokines at the same time of lymphocytes. In addition, the latest data show that compared with immunization of Indian rhesus macaques twice with Ad5 vector alone, combined immunization with adenovirus vectors of different serotypes (rAd26/rAd5) induces a stronger and broader cellular immune response. Moreover, more multifunctional CD8+ and CD4+ T lymphocytes simultaneously secreting multiple cytokines (IFN-γ/TNF-a/IL-2) were produced. More meaningfully, after intravenously infecting SIVmac251 virus, the rAd26/rAd5 combined immunization group can more effectively control the replication and infection of the virus compared with only immunizing Ad5 vector twice, and the SIVmac251 virus in this group of monkeys The peak load was reduced by 1.4 log values, and the viral load at the plateau (setpoint) was reduced by 2.4 log values. And during the whole experiment (more than 500 days), none of the monkeys in this group died of AIDS or took place in obvious AIDS symptoms, but the monkeys in other groups all died of the disease to varying degrees.

In conclusion, combined with recent research results and the failure experience of Merck vaccine, researchers in this field generally believe that the development direction of HIV-based vaccines should be able to induce stronger, broader and multifunctional T lymphocyte responses, and at the same time Induce a certain humoral immune response, so that it is possible to produce effective immune protection against HIV virus. In order to find the ultimate effective HIV vaccine, researchers should try a combination of different types of vaccines in order to find the best combination.

Adenoviral vectors have the advantages of high infection efficiency and exogenous gene expression level, simple preparation of high-titer recombinant viruses, and large capacity. Therefore, Ad vectors are favored as mammalian cell expression vectors, recombinant vaccines, and gene therapy vectors. At present, there are as many as 342 clinical trials in the world using adenovirus as a vector to treat infectious diseases, cancer, cardiovascular disease, and single-gene diseases, accounting for the first place (24.8%) among various vectors.

As a vaccine against smallpox, Tiantan poxvirus (VTT) has been used for a long time in a large number of people in China. It has very good safety and is an ideal vaccine carrier. The poxvirus vector used in this study is a further attenuated MVTT strain, which has been shown to be significantly less neurotoxic. Therefore, it can be used not only as a safe smallpox vaccine, but also as a live carrier for vaccines against other pathogens. Moreover, experiments have shown that this carrier can induce systemic mucosal reactions through mucosal routes, such as nasal cavity and oral administration.

At present, there is no report on the combined use of recombinant adenovirus vector HIV/SIV vaccine and modified poxvirus Tiantan strain (MVTT) vector HIV/SIV vaccine.

Contents of the invention

In order to develop a new type of safe and effective HIV vaccine to prevent and treat AIDS, the present invention provides a method of combined use of poxvirus vector and adenovirus vector, which can be used to prevent and treat HIV and other infectious diseases and tumors.

The method of using the poxvirus vector HIV vaccine and the adenovirus vector HIV vaccine in combination of the present invention is specifically to use the poxvirus vector HIV vaccine and the adenovirus vector HIV vaccine to immunize the body, so as to induce a more intense, broad-spectrum and multifunctional T Lymphocyte response and humoral immune response, thereby playing an effective preventive and therapeutic effect on HIV virus infection.

In the method, the pox virus vector HIV vaccine can be used to immunize the body first, and then the adenovirus vector HIV vaccine can be used to immunize the body.

In the method, an adenovirus vector HIV vaccine can be used to immunize the body first, and then a poxvirus vector HIV vaccine can be used to immunize the body.

Preferably, in the method, the poxvirus vector HIV vaccine contains HIV gag, pol, env genes.

Preferably, the poxvirus vector HIV vaccine also contains auxiliary regulatory protein genes, and the auxiliary regulatory protein genes include nef, vpx, vpr, vif, rev and tat genes.

Preferably, in the method, the adenovirus vector HIV vaccine contains HIV gag, pol, env genes.

Preferably, the adenovirus vector HIV vaccine also contains auxiliary regulatory protein genes, and the auxiliary regulatory protein genes include nef, vpx, vpr, vif, rev and tat genes.

Preferably, the way of immunizing the body refers to immunizing the body through the mucosal route, such as nasal cavity, oral cavity, etc.; immunizing the body through the intramuscular injection route; intravenous injection and all other vaccine application routes.

Preferably, the aforementioned adenovirus vector refers to any adenovirus subtype, isolate or other animal-derived adenovirus known to those skilled in the art. Specifically, it may be a type 5 or type 2 adenovirus vector.

Preferably, the aforementioned poxvirus vector refers to any poxvirus subtype, isolate known to those skilled in the art. Specifically, it may be a modified Tiantan strain poxvirus vector.

The present invention also provides the application of the above-mentioned method of using poxvirus vector and adenovirus vector in combination in the field of preventive and therapeutic HIV vaccines to prevent and treat AIDS, and to prevent and control hepatitis B, Ebola virus and tumors .

Compared with HIV vaccines using poxvirus vectors or adenovirus vectors alone, combined use of poxvirus vectors and adenovirus vectors carrying HIV gag, pol, and env genes induced stronger, broader and multifunctional T Lymphocyte response, and showed a strong protective and control effect on rectal infection of SIVmac239 virus. Data have shown that strong, broad-spectrum and multifunctional T lymphocyte responses, especially killer T lymphocyte responses, play a very important role in controlling various infectious viral infectious diseases and tumors, so in the present invention The strategy of enhancing the body to produce a stronger, broader and multifunctional T lymphocyte response is also suitable for preventing and treating various viral infectious diseases, such as hepatitis B, Ebola virus, etc., as well as various tumors. In addition, since poxvirus vectors and adenovirus vectors have been widely used in clinical trials, they have good safety profiles, making this strategy very promising for clinical application. The inventor's experimental data has laid a solid foundation for this vaccine strategy to enter clinical trials. The invention can be generally used in the prevention and treatment of AIDS, as well as the prevention and control of other infectious diseases and tumors.

The present invention will be further described below in conjunction with drawings and embodiments.

Description of drawings

Fig. 1 is the detection result figure that detects recombinant adenovirus protein expression level with western-blot method;

Figure 2 is a comparison of the immunogenicity of the recombinant adenovirus in mice after the initial immunization and booster immunization; Figure 2A is a comparison of the immunogenicity of the recombinant adenovirus in mice after the initial immunization; Figure 2B is a comparison of the immunogenicity of the recombinant adenovirus in mice Comparison of immunogenicity of the virus after booster immunization in mice;

Fig. 3 is the immunization and challenge program in rhesus macaques of using poxvirus vector (MVTT) SIV vaccine and adenovirus (Ad5) vector SIV vaccine in combination;

Figure 4 is the immunogenicity of ELISPOT detection combined with MVTT vector and Ad5 vector in rhesus monkeys; Figure 4A is the ELISPOT detection data 6 weeks from the last immunization, and Figure 4B is the ELISPOT detection data 21 weeks from the last immunization;

Figure 5 is the result of detecting cytokines secreted by Gag peptide-stimulated CD8+ T cells by multi-color flow cytometry 6 weeks after rhesus macaques were boosted with MVTT and Ad5 vector HIV vaccine;

Figure 6 is the combination of MVTT and Ad5 carrier HIV vaccine to rhesus macaques for 16 weeks after the booster immunization, and the detection results of memory T cells secreting cytokines in peripheral blood detected by multi-color flow cytometry; Figure 6A is the detection effect The experimental data of CD8 T cells secreting various cytokines; Figure 6B is the experimental data of detecting central CD8 T cells secreting various cytokines;

Figure 7 is the results of the detection of the proliferation ability of T lymphocytes by staining with the living cell dye carboxyfluorescein acetoacetate succinimidyl ester (CFSE) 16 weeks after MVTT combined with the Ad5 vector HIV vaccine to rhesus macaques for booster immunization ; Figure 7A is the proliferation of CD4 T cells; Figure 7B is the proliferation of CD8 T cells;

Fig. 8 is the cellular immune response against each antigen of SIV in the experimental monkey after being challenged with SIVmac239; Fig. 8A is the immune response against the structural proteins (Gag, Pol and Env); Fig. 8B is the immune response against the accessory regulatory proteins (Nef, Vpx, Vpr, Vif, Rev and Tat) immune response;

Figure 9 is the detection of SIV-specific antibody response levels before and after SIVmac239 infection in experimental monkeys by ELISA technology;

Fig. 10 is a graph showing the data results of detecting the peak viral load in the experimental monkey by quantitative PCR technology.

Detailed ways

In order to make the present invention easier to understand, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. 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 vaccines carrying SIVgag, pol and env genes and immunogenicity in mice

1. Acquisition of SIVmac239 gene

Each gene required for the experiment was obtained by total gene synthesis. This embodiment involves 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 the amino acid sequences, so that these antigens were highly expressed in primate cells.

1.1 The optimized sequence of the SIVmac239gag gene used in this embodiment 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 cacagtgtgtgtgatctggt 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 ctgcacccccc 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 embodiment 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 cctacaccccc 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

tccatggcca ggccaactct gacctgggca cctggcagat ggactgcacc catctggagg 2520

gcaagatcat cattgtggct gtgcatgtgg cctctggctt cattgaggct gaggtgatcc 2580

cccaggagac aggcaggcag acagccctgt tcctgctgaa gctggctggc aggtggccca 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 tgcactgcat gaacttcaag aggaggggcg 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 caacaacaca 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

agaccaacat 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 gagc 2684

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

According to the conventional recombinant adenovirus construction method, the above genes were respectively inserted into the adenovirus vector, wherein the adenovirus vector was a human type five adenovirus with deletion of the E1 and E3 regions, and then the recombinant adenovirus carrying the target gene was rescued, and a small amount of amplification was obtained. After amplification, expression and genomic restriction identification were carried out, followed by massive amplification in Trex293 cells, and purification by cesium chloride gradient density centrifugation. After the purified virus was identified correctly again, its infectious titer TCID ₅₀ and physical particle concentration were measured. Then subpackage and freeze for future use (for the specific operation steps of related methods, please refer to the applicant's patent, application number is 200710026295.X). Figure 1 is a diagram of the detection results of recombinant adenovirus protein expression levels detected by western-blot method; as shown in Figure 1, the purified recombinant adenovirus can express the target protein at a high level, and these proteins can be further processed into natural forms Various protein components. For example, Ad5-gag can express P55 and P27, Ad5-pol can express P66/51, P31, P10, etc., Ad5-env can express gp160, gp120, gp41.

3. Immunogenicity of SIV full antigen group vaccine carrying adenovirus vector in mice

Further, the immunogenicity of these vaccines was verified in mice, with 10 6-8-year-old female C57BL/6 mice per group. The immunization dose was 1×10 ¹⁰ vp recombinant adenovirus/mouse/100 μL, and 50 μL was injected into the quadriceps femoris on both sides of the mouse. A total of 2 immunizations with an interval of 2 weeks. Figure 2 is a comparison of the immunogenicity of the recombinant adenovirus in mice after the initial immunization and booster immunization; Figure 2A is a comparison of the immunogenicity of the recombinant adenovirus in mice after the initial immunization; Figure 2B is a comparison of the immunogenicity of the recombinant adenovirus in mice Comparison of immunogenicity of the virus after booster immunization in mice; as shown in Figure 2A and 2B, the spleens of the mice were taken out after the initial immunization and booster immunization, mixed together by group, and spleen cells were prepared for ELISPOT detection. In each immunization group, strong IFN-γ ELISPOT immune response could be detected in mice, and the response level against SIV Gag and Pol was relatively higher. More meaningfully, in the fifth group, that is, the mice immunized with SIVgag, pol and env at the same time can generate immune responses against these three SIV antigens at the same time. Compared with immunization with one antigen alone, the level of immune response in this group decreased, which may be related to the simultaneous high-level expression of multiple antigens in the body, which will lead to competition between antigens. Although the response to a single antigen has decreased, we can also see that the overall response level to each antigen is more consistent, that is, the response to each antigen is more coordinated, and the excessive response to some antigens is avoided, which may be It is more conducive to controlling the replication of SIV virus. No immune response against any of the SIV antigens was detectable in control mice.

The poxvirus vector MVTT-gpe (improved poxvirus Tiantan strain) expressing SIVgag, pol and env was constructed and provided by Professor Chen Zhiwei, Institute of AIDS, Li Ka Shing Faculty of Medicine, University of Hong Kong, and their expression and antigenicity were confirmed by them (Huang X et al. Vaccine. 2007).

Example 2: Combined use of poxvirus vector SIV vaccine and adenovirus vector SIV vaccine immune response in rhesus monkeys

1. Experimental materials

1.1 Experimental animals

16 Chinese rhesus monkeys were purchased from the non-human primate disease model research base jointly established by the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences and the South China Institute of Endangered Animals, with a weight of 3-5kg and an age of 3-6 years. Male and female. Various tests have been carried out on these monkeys before the experiment, and it is confirmed that the tuberculosis, Shigella, Salmonella, STLV-1, SIV, SRV, etc. are all negative. The operation of animal experiments complied with the relevant regulations on the use of experimental animals.

1.2 Viruses

The poxvirus 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 of the University of Hong Kong; the recombinant adenovirus (Ad5-SIVgag, Ad5-SIVpol and Ad5-SIVpol and Ad5-SIVenv).

1.3 Flow Antibody

Monoclonal antibodies used (anti-human CD3-pacific blue (Pacific Blue), anti-NHP CD4-FITC, anti-human CD4-AmCyan, anti-human CD8-PerCP, anti-human CD8-APC-Cy7 (SK1), anti-human CD28-FITC , anti-human CD95-PE-Cy5, anti-human TNFa-PE-CY7, anti-human IFNγ-PE, anti-human IL2-APC) were purchased from BD Company.

1.4 Peptides of each antigen of SIV

All peptides are provided by the NIHAIDS Research & Reference Reagent Program of the National Institutes of Health. Most of the peptides consist of 15 amino acids, with 11 amino acid overlaps between each pair. The purity is >80%. It was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution of 0.4 mg/ml/peptide, and stored at -70°C after aliquoting.

1.5 Other reagents

ELISPOT plate (brand: Millipore; product number: MSIPS4510) was purchased from Dakowei Biotechnology Co., Ltd., SIV virus particle lysate was prepared by ourselves, and TMB/E substrate (product number: ES001) was purchased from Chemicon, USA. BCIP/NBT substrate (brand: Pierce; product number: 34042) was purchased from Guangzhou Jitai Biotechnology Co., Ltd. The cell dye CFSE was purchased from Molecule Probe. Streptomycin-conjugated alkaline phosphatase (brand: BD PharMingen, product number: 554065) was purchased from Gene Co., Ltd.

2. Buying inspection method

2.1 Animal grouping and immunization strategy

Divide 16 monkeys into 4 groups, 4 monkeys in each group, as shown in Figure 3, the immunization and challenge scheme in rhesus monkeys using poxvirus vector (MVTT) SIV vaccine and adenovirus (Ad5) vector SIV vaccine . Detection indicators include ELISPOT, multifunctional T cells, cell proliferation and antibody titer, etc. The adenovirus was immunized by intramuscular injection, and the dose was 10 ¹¹ vp/monkey. The dose of pox virus was 1×10 ⁸ PFU/ml/monkey, and nasal cavity and sublingual immunization were adopted.

2.2 Detection of multifunctional T cells by multi-color flow cytometry

1) The separated PBMC cells were counted and adjusted to 2×10 ⁶ /ml, and added to a 24-well culture plate, 1 mL per well. The culture medium is R10.

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

3) Incubate at 37°C, 5% CO ₂ for 1-2 hours.

4) Add brefeldin (BFA) (10 μg/ml) and mix well.

5) Cultivate at 37°C, 5% CO ₂ for 16 hours.

6) Pipette the cells evenly and transfer them to flow tubes (12×75mm polystyrene tubes). Add 3ml flow wash solution. After mixing, centrifuge at 300g room temperature for 10 minutes.

7) Discard the supernatant. Add cell-specific labeled antibodies, such as CD3-pacific blue (Pacific blue), CD4-AmCyan, CD8-APC-Cy7, CD28-FITC, CD95-PE-Cy5, and shake for 2-3 seconds.

8) Store at room temperature in the dark for 25-30 minutes.

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

10) Repeat the wash once.

11) Shake the cells evenly, then add 250 μl cell fixation/permeabilization solution (cytofix/cytoperm, brand: BD; product number: 554722), and mix well.

12) React at 4°C for 20 minutes in the dark.

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

14) Repeat once.

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

16) Protect from light at 4°C for 30-60 minutes.

17) Add 1 ml of 1× cell permeation/wash solution (perm/wash), 400 g, and centrifuge for 8 min.

18) Discard the supernatant, and add 3ml flow washing solution. After mixing, centrifuge at 400 g for 10 minutes at room temperature.

19) Suck off most of the supernatant, leaving about 300 μl. Shake and mix well before going on the machine.

20) Detect on a FACSAria flow cytometer, acquire data with Cell Quest software, and then analyze with Modfit software.

2.3 IFN-γ ELISpot technology

first day:

Take a tube of coating antibody (the anti-monkey IFN-γ antibody pair contains this antibody, brand: U-Cytech, clone number is MD-1) and dilute it with sterile PBS (pH7.2-7.4) 1:100, 100μl Each well was added to a 96-well PVDF membrane plate and coated overnight at 4°C.

the next day:

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

②PBMCs were isolated during the sealing period. Lymphocytes were separated using 95% diluted lymphocyte separation medium (trade name: opti-prep).

③ Discard the blocking solution, and add 100 μl PBMC to each well.

④ A positive control group (ConA group), a specific peptide, and a blank control (DMSO) were set up in the experiment. Incubate at 37°C in a 5% CO2 incubator.

third day:

⑤After 24 hours, discard the cell suspension and wash 6 times with sterile PBST 220μl/well.

⑥ Shake off the lotion, and dry it on a sterile desiccant paper.

⑦ Dilute the detection antibody (biotin-labeled anti-monkey IFN-γ antibody) 1:400 with PBST containing 5% FBS, 100 μl/well, overnight at 4°C.

fourth day:

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

② Shake off the lotion, and dry it on a sterile desiccant paper.

③ Prepare streptomycin-coupled alkaline phosphatase: dilute streptomycin-coupled alkaline phosphatase with PBST containing 5% FBS at 1:2500, 100 μl/well, 37°C, 2 hours.

④ Treat BCIP/NBT substrate (Pierce, Cat: 34042), incubate at 37°C for 30min.

⑤ Wash 5 times with sterile PBST 220 μl/well, shake off the washing solution, and dry it on sterile dry paper.

⑥ Add BCIP/NBT substrate, 100 μl/well, and react in the dark for 7 minutes.

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

2.4 CFSE detection of lymphocyte proliferation

1) Isolate the PBMC cells of rhesus monkeys.

2) After cell counting, centrifuge at 300 g for 10 minutes. Discard the supernatant, add 0.1% FBS/PBS liquid to resuspend, wash once, and then resuspend with an appropriate volume of 0.1% FBS/PBS to obtain a cell suspension of 4×10 ⁶ /ml.

3) Dilute the CFSE stock solution to 2 μmol/L with 0.1% FBS/PBS. The following operations should be done away from light as much as possible.

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

5) Place at 37°C for 10 minutes.

6) Add ice-cold RPMI 1640 complete culture solution, ice bath for 5 minutes to stop staining.

7) Centrifuge at 300g room temperature for 5 minutes. Discard the supernatant, add ice-cold complete 1640 culture solution containing 10% FBS and wash twice. Finally, resuspend with 1ml R10 medium.

8) Add the above cell suspension into a 24-well culture plate, 1ml per well, and stimulate with SEB (final concentration 1ug/ml) and specific peptide library (2μg/ml) respectively.

9) Culture at 37° C., 5% CO2 in the dark for 5-6 days.

10) Pipette the cells evenly and transfer them to flow tubes (12×75mm polystyrene tubes). Then add 4ml FACS wash solution. After mixing, centrifuge at 250 g for 10 minutes at room temperature.

11) Vacuum pump to suck off the supernatant. Vortex for 2-3 seconds.

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

13) Store at room temperature in the dark for 25-30 minutes.

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

15) Suck away most of the supernatant, leaving about 300ul. Vortex to mix. Go straight to the machine. Or continue to step 15.

16) Add 30 ul of 10% formalin (brand: Polysciences; product number: 04018), and shake for 10-20 seconds.

17) Store at 4°C in the dark, and test on the machine within 24 hours.

18) Detect on a FACScalibur flow cytometer, acquire data with Cell Quest software, and then analyze with Modfit software.

2.5 Data analysis

Multicolor flow cytometry data were analyzed using FlowJo software. JMP version 6.0.3 software was used for statistical analysis and graphing of data, and non-parametric Wilcoxon rank test was used to compare the differences between groups.

3. Experimental results

3.1 MVTT/Ad5 combined immunization strategy induces a stronger and more durable cellular immune response in Chinese rhesus monkeys

We compared the immunogenicity of using MVTT vector and Ad5 vector alone or in combination in monkeys, and the results showed that the immune response induced by a single immunization with Ad5 vector was much higher than that induced by MVTT vector, while the immune effect of combining these two vectors was It is far stronger than using any kind of carrier alone. Figure 4 is the ELISPOT detection of the immunogenicity of combined use of MVTT vector and Ad5 vector in rhesus monkeys; Figure 4A is the ELISPOT data 6 weeks from the last immunization, and Figure 4B is the ELISPOT data 21 weeks from the last immunization. As shown in Figure 4, we observed that a single immunization with the modified poxvirus Tiantan strain (MVTT) vector induced a certain intensity of cellular immune response in monkeys, and the stimulation of the target antigen carried by it produced an average of 237 spots ( millions of PBMC cells); a single immunization of the type 5 adenovirus vector we constructed produced a strong cellular immune response in monkeys, and the stimulation of the target antigen carried by it produced an average of 2643 spots (millions of PBMC cells). However, the MVTT carrier was used for primary immunization, and after an interval of 6 weeks, it was strengthened with Ad5 carrier, which induced a very high level of cellular immune response in monkeys, and the stimulation of the target antigen carried by it produced an average of 7383 spots (millions of PBMCs). cell). Therefore, this strategy is 31 times stronger on average than the response level of cellular immunity induced by a single MVTT vector, and 2.8 times stronger than that of an Ad5 vector alone. This strategy not only quickly induced a very high level of cellular immune response in monkeys, but more importantly, this response lasted for a long time. A high level of ELISPOT response could still be detected in monkeys in the combined immunization group 21 weeks after the last immunization.

3.2 MVTT/Ad5 combined immunization strategy induced more multifunctional memory lymphocytes in Chinese rhesus monkeys

Although IFN-γELISPOT technology has become a more general and widely recognized index for evaluating HIV vaccines, the more recognized view today is that immune protection is more correlated with the number of lymphocytes with multiple functions, so-called multifunctional lymphocytes. Cells refer to lymphocytes that simultaneously secrete various cytokines including IFN-γ, including IL-2, TNFa, MIP1β, etc. Evaluation of multifunctional T lymphocytes will gradually become an important indicator for evaluating the effect of HIV vaccines. Therefore, we have developed and improved multicolor flow cytometry to measure the response of multifunctional T lymphocytes produced by this strategy. The results are shown in Figure 5. Figure 5 is the result of multi-color flow cytometry detection of cytokines secreted by Gag peptide-stimulated CD8+ T cells 6 weeks after MVTT combined with Ad5 vector to boost immunization of rhesus monkeys; in MVTT After 6 weeks of co-immunization with Ad5 vector, not only the ability to secrete a certain cytokine alone was greatly enhanced in the monkeys in the co-immunization group, but more importantly, more cells secreting IFN-γ/TNF-a/IL-2 at the same time were detected multifunctional CD8+ T lymphocytes. Specifically, in monkeys co-immunized with MVTT and Ad5 vector, 0.2464±0.2770% of CD8+ T lymphocytes simultaneously secreted the three cytokines IFN-γ/TNF-α/IL-2, and 1.4668±1.1845% of CD8+ T lymphocytes secreted IFN-γ/TNF-α at the same time; while only 0.0320±0.0472% of CD8+ T lymphocytes in monkeys immunized with Ad5 vector alone secreted IFN-γ/TNF-α/IL-2 Of these three cytokines, 0.1587±0.2035% of CD8+ T lymphocytes secreted IFN-γ/TNF-α simultaneously. That is, compared with Ad5 vector immunization alone, the combined immunization of MVTT and Ad5 vector produced 7.7 times more multifunctional CD8+ T lymphocytes and There are 9 times more multifunctional CD8+ T lymphocytes that can simultaneously secrete two cytokines, IFN-γ/TNF-α.

We further tested the ability of specific memory cells to produce and secrete cytokines after co-immunization with MVTT and Ad5 vector. The results are shown in Figure 6. Figure 6 is the result of detecting cytokines secreted by memory T cells in peripheral blood using multi-color flow cytometry 16 weeks after MVTT and Ad5 vectors were combined to boost immunization in rhesus monkeys; 6A is the experimental data for detecting the secretion of various cytokines by effector CD8 T cells; FIG. 6B is the experimental data for detecting the secretion of various cytokines by central CD8 T cells. Both SIV antigen-specific memory CD4+ T cells and CD8+ T cells in these monkeys were significantly more capable of producing and secreting cytokines. Compared with other groups, the ability of effector memory CD8+ cells in the peripheral blood of monkeys in the MVTT and Ad5 combined immunization group to secrete cytokines was significantly stronger, and these cells mainly secreted IFN-γ/TNF-a two cytokines at the same time . Although there was little difference in the ability of the central memory CD8+ cells to secrete cytokines among the experimental monkeys in each group, the ability of these cells to secrete IL-2 was significantly stronger than that of the effector memory T cells. In summary, we detected that combined immunization with MVTT and Ad5 can induce more multifunctional memory T lymphocytes in rhesus monkeys. Further analysis showed that this method simultaneously induced central memory and effector memory T cells, among which effector memory CD8+ T cells were the main ones.

3.3MVTT/Ad5 combined immunization strategy induced stronger lymphocyte proliferation in Chinese rhesus monkeys

Studies have shown that CD4+T cells and CD8+T cells of patients with long-term HIV non-progression have a strong ability to proliferate against HIV antigens in vitro, while CD4+T cells and CD8+T cells of HIV-onset patients can proliferate in vitro against HIV antigens. Very weak. Moreover, there is a certain degree of negative correlation between this proliferation ability and the virus copy number in the patient's plasma, and a positive correlation with the number of CD4+ T cells. Therefore, detecting the proliferative ability of T lymphocytes can better reflect the degree of control of the body on HIV virus replication.

Figure 7 is the result of CFSE staining to detect the proliferation ability of T lymphocytes after 16 weeks of booster immunization of rhesus macaques in combination with MVTT and Ad5 vector; Figure 7A is the proliferation of CD4 T cells; Figure 7B is the proliferation of CD8 T cells Condition. As shown in Figure 7A, after stimulation with SIV Gag in PBMCs of monkeys immunized with MVTT and Ad5 vectors in vitro, (4.2725±4.5516)% of CD4+ cells were CFSElow, that is, progeny cells of division and proliferation; while those of monkeys immunized with Ad5 vector alone After PBMC were stimulated with SIV Gag, (3.2350±1.7008)% of CD4+T cells were CFSElow. Therefore, compared with Ad5 vector alone, the proliferation ability of CD4+ T lymphocytes stimulated by antigen was slightly enhanced after combined immunization with MVTT and Ad5 vector. As shown in Figure 7B, after being stimulated with SIV Gag, (7.4825±5.8550)% of CD8+T cells were CFSElow, that is, progeny cells of division and proliferation; while only (2.9475±1.6951)% of PBMCs of monkeys immunized with Ad5 vector alone % of CD8+ T cells were CFSElow. Therefore, compared with the Ad5 vector alone, the proliferation ability of CD8+ T lymphocytes stimulated by antigen was enhanced by more than 2.5 times after MVTT and Ad5 vector co-immunization.

In conclusion, MVTT vector priming and Ad5 vector boosting immunization method produced stronger, broader and more functional T cell responses in Chinese rhesus macaques, which is very consistent with the indicators recently evaluated for effective vaccines. This strategy deserves further study.

Example 3: Immunological protection test in rhesus macaques using poxvirus vector and adenovirus vector SIV vaccine in combination

1 Experimental materials:

1.1 Experimental animals are the same as in Example 2.

1.2 Viruses

The SIVmac239 virus was rescued by the inventor, and the SIVmac239phage molecular clone used was provided by the AIDS Research and Reference Reagent Project of the National Institute of Health of the United States.

1.3 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(SK1), anti-human CD28-FITC, anti-human CD95 -PE-Cy5, anti-human TNFa-PE-CY7, anti-human IFNγ-PE, anti-human IL2-APC) were purchased from BD Company. HRP-anti-monkey IgG was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

1.4 Other reagents

Fluorescent quantitative RT-PCR detection kit (QuantiTect SYBR Green RT-PCR Kit, catalog number: 204243) was purchased from Qiagen, red blood cell lysate (FACS lysis solution), absolute counter tubes (BDTruCOUNT Tubes, catalog number 340334) were purchased from BD Biosciences company. See Example 2 for other reagents.

2 Experimental methods

2.1 Animal grouping and challenge experiment

The 16 monkeys were divided into 4 groups, 4 monkeys in each group. The immunization program of each group was shown in Figure 3. The rectum was infected with SIVmac239 virus 22 weeks after the last immunization, and the dose was 10 ⁵ TCID ₅₀ . The specific method is to fast the monkeys for 24 hours, keep the buttocks high, insert the anus 4-7cm with a gastric tube, inject the virus, and inject a small amount of air. Maintain this position for at least 20 minutes.

2.2 Quantitative PCR determination of SIV viral load

1) Plasma was separated by conventional methods.

2) extract the SIV virus RNA in the plasma.

3) Perform one-step fluorescent quantitative RT-CR, and make a standard curve for each test.

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

2x QuantiTect SYBR Green RT-PCR master reaction solution 25μl

QuantiTect RT mix 0.5μl

The kit comes with primer 1 (10μM) 0.5μl

The kit comes with primer 2 (10μM) 0.5μl

RNA template 2 μl

RNase-free water 21.5μl

Total volume 50μl

5) Reasonably arrange the test layout according to the number of samples. Then carry out RT-PCR reaction conditions according to the following conditions:

a) 50℃ 30min

b) 95℃ for 15 minutes

c) 94°C 15s

d)60℃ 30s

e) 72°C 30s

f) plate reading;

g) 3)-6) cycle for 45 weeks;

h) Make a melting curve at 65-95 degrees, and keep reading for 1 second every 0.5 degrees;

i) kept at 24°C;

6) According to the standard curve, use Opticon Monitor 3 software to obtain the virus copy number of each sample, and then restore the virus copy number per milliliter of blood according to the dilution factor.

2.3 SIV virus particle lysate ELISA method to detect binding antibody

1) Antigen coating: 100 μl antigen (1 μg/ml, diluted with PBS pH 7.4 or carbonate buffer solution pH 9.6) was added dropwise to each well of a 96-well plate, and coated overnight at 4°C.

2) Plate washing: pour off the antigen the next day, spin dry, and wash with PBS 3 times.

3) Blocking: add PBST containing 5% skimmed milk powder, 250 μl per well, block at 37° C. for 1 h.

4) Plate washing: add 200 μl PBST to each well, wash 3 times for 3 min/time, and keep it moist for later use.

5) Add the sample to be tested: 100 μl is added to each well, and each sample is measured in two wells, and reacted at 37° C. for 2 hours. (if diluted, use blocking solution)

6) Plate washing: add 200 μl PBST to each well, wash 6 times for 3 min/time.

7) Binding to the second antibody: add 100 μl of appropriate dilution HRP-anti-mouse IgG (1:8000) antibody or anti-human IgG (1:4000) to each well, and incubate at 37° C. for 1 hour. (dilute with blocking solution)

8) Plate washing: add 200 μl PBST to each well, wash 6 times for 3 min/time.

9) Color development: add 100 μl enzyme reaction substrate TMB to each well, and let stand at room temperature for 5-10 minutes.

10) Add an equal volume of 1M H ₂ SO ₄ to stop the reaction.

11) Determination of results: Measure the OD ₄₅₀ value at a wavelength of 450nm with a microtiter plate spectrophotometer

2.4 Refer to Example 2 for other methods.

3. Experimental results

3.1 Cellular immune responses against various antigens in rhesus monkeys after challenge

Fig. 8 is the cellular immune response against each antigen of SIV in the experimental monkey after being challenged with SIVmac239; Fig. 8A is the immune response against the structural proteins (Gag, Pol and Env); Fig. 8B is the immune response against the accessory regulatory proteins (Nef, Vpx, Vpr, Vif, Rev and Tat) immune response. As shown in Figure 8A, after being challenged by SIVmac239 virus, the monkeys in the combined immunization vaccine group had a rapid increase in the response to the immunogen (gag/pol/env) carried by the vaccine, while the response in the control group was relatively slow, and the response The strength is also relatively weak. This shows that the immunization method of the present invention effectively induces memory T cells, and these cells are rapidly activated and generate a corresponding immune response after being stimulated by the same antigen. What is more interesting is that the response to the immunogen (nef/vpx/vpr/vif/rev/tat) not carried by the vaccine in the monkeys of the combined immunization group was significantly weaker than that of the control group. We speculate that this is due to the presence of SIV in these monkeys. The body is effectively controlled at a relatively low level or is not infected, so the level of response to the antigen carried by the virus itself is relatively low. This guess was confirmed in the following viral load assay experiments.

3.2 Antibody immune response to SIV in rhesus monkeys after challenge

We also detected the antibodies in the serum of these experimental monkeys in different periods. As shown in Figure 9, the SIV-specific antibody response levels before and after SIVmac239 infection in the experimental monkeys were detected by ELISA technology. Antibody levels in most monkeys were low, but antibody levels rose rapidly in most monkeys after the booster immunization. More significantly, the antibody levels of monkeys in the immunized group rose rapidly to very high levels after being challenged by the virus. The experimental monkeys in the control group could not detect the antibody 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

Viral loads in all monkeys peaked 10-14 days after challenge. It can be seen from Figure 10 that the peak viral load in monkeys in the combined immunization group was significantly lower than that in the control group. One of the four monkeys in the joint immunization group has not detected SIV virus so far, and the viral load of the other three monkeys has decreased by 1.37log compared with the control group. This shows that our combined strategy effectively controls SIV virus infection and replication during the acute infection period, and the long-term immune protection effect of this method needs to be confirmed by longer observation time. One thing to note is that we used the more difficult-to-neutralize SIVmac239 strain to infect the mucosa with high doses, while most of the previous studies used relatively weaker strains.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

sequence listing

<120> Method and application of combined use of poxvirus vector HIV vaccine and adenovirus vector HIV vaccine

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agaagatcag gctgaggccc aatggcaaga agaagtacat gctgaagcat gtggtgtggg 120

ctgccaatga gctggacagg tttggcctgg ctgagtccct gctggagaac aaggagggct 180

gccagaagat cctgtctgtg ctggcccccc tggtgcccac aggctctgag aacctgaagt 240

ccctgtacaa cacagtgtgtgtgatctggt 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 ctgcacccccc 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

<210>1

<211>3227

<212> DNA

<213> SIVmac239pol

<222>(1)...(3227)

<400>2

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 cctacaccccc 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

tccatggcca ggccaactct gacctgggca cctggcagat ggactgcacc catctggagg 2520

gcaagatcat cattgtggct gtgcatgtgg cctctggctt cattgaggct gaggtgatcc 2580

cccaggagac aggcaggcag acagccctgt tcctgctgaa gctggctggc aggtggccca 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 tgcactgcat gaacttcaag aggaggggcg 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

<210>1

<211>2684

<212>DNA

<213> SIVmac239env

<222>(1)...(2684)

<400>3

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 caacaacaca 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

agaccaacat 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 gagc 2684

Claims (12)

1. The method of using the poxvirus vector HIV vaccine and the adenovirus vector HIV vaccine in combination, which is characterized in that the poxvirus vector HIV vaccine and the adenovirus vector HIV vaccine are used in combination to immunize the body. 2. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that the poxvirus vector HIV vaccine is used to immunize the body first, and then the adenovirus vector HIV vaccine is used to immunize the body. 3. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that the adenovirus vector HIV vaccine is used to immunize the body first, and then the poxvirus vector HIV vaccine is used to immunize the body. 4. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that said poxvirus vector HIV vaccine contains HIV gag, pol and env genes. 5. The method for using the poxvirus vector HIV vaccine and the adenovirus vector HIV vaccine in combination according to claim 4, characterized in that, the poxvirus vector HIV vaccine can also contain an auxiliary regulatory protein gene, and the auxiliary regulatory protein gene Genes include the nef, vpx, vpr, vif, rev, and tat genes. 6. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that said adenovirus vector HIV vaccine contains HIV gag, pol and env genes. 7. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 6, characterized in that, the adenovirus vector HIV vaccine can also contain an auxiliary regulatory protein gene, and the auxiliary regulatory protein Genes include the nef, vpx, vpr, vif, rev, and tat genes. 8. The method of using poxvirus-vectored HIV vaccine and adenovirus-vectored HIV vaccine in combination according to claim 1, characterized in that the ways of immunizing the body include mucosal way, intramuscular injection way and intravenous way. 9. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that the adenovirus vector is type 5 adenovirus vector. 10. The method of using poxvirus vector HIV vaccine and adenovirus vector HIV vaccine in combination according to claim 1, characterized in that said poxvirus vector is a modified Tiantan strain poxvirus vector. 11. The application of the method of combined use of poxvirus vector HIV vaccine and adenovirus vector HIV vaccine according to any one of claims 1-10 in the prevention and treatment of AIDS. 12. The application of the combined use of poxvirus vector HIV vaccine and adenovirus vector HIV vaccine according to any one of claims 1-10 in the prevention and control of hepatitis B, Ebola virus and tumors.

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