WO2006079290A1 - A recombinant sars-cov vaccine comprising attenuated vaccinia virus carriers - Google Patents

A recombinant sars-cov vaccine comprising attenuated vaccinia virus carriers Download PDF

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WO2006079290A1
WO2006079290A1 PCT/CN2006/000181 CN2006000181W WO2006079290A1 WO 2006079290 A1 WO2006079290 A1 WO 2006079290A1 CN 2006000181 W CN2006000181 W CN 2006000181W WO 2006079290 A1 WO2006079290 A1 WO 2006079290A1
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sars
mva
vaccine
cov
ads
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Chuan Qin
Qiang Wei
Hong Gao
Xinming Tu
Zhiwei Chen
Linqi Zhang
David D. Ho
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Institute Of Laboratory Animal Science Of Chinese Academy Of Medical Sciences
The Aaron Diamond Aids Research Center
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Abstract

The present invention discloses a recombinant SARS-CoV vaccine (ADS-MVA) comprising attenuated vaccinia virus (MVA) carriers for preventing and curing SARS. We found that the recombinant ADS-MVA has good immunogenicity, reliability and effectiveness by the determination of immunogenicity, reliability and effectiveness of the vaccine, the determination of antibody, the dynamic detection of viral load in vivo and the ascertainment of index of the change of SARS pathological degree. It is a perfect vaccine for preventing and curing SARS.

Description

 Recombinant vaccinia-SARS vaccine and preparation method thereof

 The present invention relates to a recombinant vaccinia-SARS vaccine and a preparation method thereof, and more particularly to a SARS vaccine ADS-MVA comprising a modified attenuated vaccinia MVA and a SARS coronavirus S gene and a preparation method thereof.

Background technique

 Since SARS was first discovered and reported in Guangdong Province in November 2002, it has quickly become popular worldwide, and WHO calls it Severe Acute Respiratory Syndrome (SARS). The disease is mainly transmitted by close-range airborne locusts and close contact, which is characterized by the general population's susceptibility, rapid spread, acute onset and certain mortality. At present, there are 5,663 confirmed cases and suspected cases of SARS in the world. As of May 15, 2004, there were 372 deaths. As of May 26, 2004, there were 5,316 cases, 315 deaths, 1573 cases were suspected, and 2,742 cases were cured. . The widespread prevalence of SARS is a serious threat to human health. Studies at home and abroad have shown that the SARS pathogen is a variant coronavirus with a positive long-chain RNA of 29742 bp.

 China has made some progress in the study of SARS pathogens, diagnosis, and sources. In our previous work, animal models of SARS infection have been established. At the beginning of May 2003, we were infected with rhesus monkeys, systematically conducting virology, serology, and pathology studies. It was important to detect high-valency neutralizing antibodies in infected monkeys. This result confirmed the SARS virus. It can induce the production of protective antibodies in the body. Therefore, it is theoretically possible to develop a SARS virus vaccine. The infected monkey is reliable as a model animal for vaccine evaluation. This work has solved the restriction of drug screening, vaccine evaluation, etc. for a considerable period of time. The bottleneck of the research, especially for the research of vaccine evaluation, has laid an important foundation. The complete control of the disease depends on the development of effective vaccine.

 A SARS vaccine comprising a SARS-associated coronavirus S gene and a eukaryotic expression plasmid is disclosed in Chinese Application No. 200410044291.0, and a SARS vaccine comprising an adenovirus vector and a SARS-associated coronavirus S gene is disclosed in Chinese Patent Application No. 200410044285.5, but the existing SARS There are different levels of safety hazards in vaccines, and their effectiveness remains to be further observed and validated.

SUMMARY OF THE INVENTION

Existing SARS vaccines The present invention overcomes many existing technical problems and successfully develops A recombinant vaccinia-SARS vaccine with good immunogenicity, safety and efficacy, and the use of the vaccine to prevent pathogenic SARS-CoV infection in the animal model of Chinese rhesus monkey, has achieved satisfactory results. Especially compared with the inactivated SARS vaccine in clinical trials, this vaccine is safer and more reliable without contact with SARS-CoV.

 A first aspect of the invention relates to a SARS-CoV vaccine (ADS-MVA) constructed on the basis of attenuated vaccinia virus MVA.

 The above SARS-CoV vaccine was obtained by inserting the SARS-associated coronavirus S gene into the MVA deletion III region.

 The invention also relates to a method for preparing a SARS-CoV vaccine, comprising:

 (1) constructing a shuttle plasmid comprising a SARS-CoV antigen gene, and an attenuated vaccinia virus MVA deletion region III gene coding region sequence or a flanking sequence homologous to a sequence flanking the coding region;

(2) homologous recombination of the shuttle plasmid with the attenuated vaccinia virus MVA strain in the permissive cells of the vaccinia virus;

 (3) The recombinant virus was subjected to single spot screening by screening markers to isolate a pure highly attenuated vaccinia ADS-MVA virus.

 The present invention also relates to a SARS-CoV vaccine (ADS-MVA) prepared by the above-described SARS-CoV vaccine preparation method.

 Further, the present invention relates to the use of the aforementioned attenuated recombinant vaccine SARS-CoV vaccine ADS-MVA for the treatment or prevention of SARS-CoV infection and disease.

 The present invention relates to the use of the aforementioned method for constructing attenuated recombinant vaccinia SARS vaccine ADS-MVA for constructing recombinant MVA for expression of a foreign gene.

 The invention further relates to a method for constructing the aforementioned attenuated recombinant vaccinia SARS vaccine ADS-MVA for use in the construction of a recombinant MVA expressing a foreign pathogen antigen for the preparation of a pathogen detection and diagnostic reagent.

Furthermore, the present invention relates to a method for preventing pathogenic SARS-CoV infection in a Chinese rhesus monkey by the aforementioned attenuated recombinant vaccinia SARS-CoV vaccine ADS-MVA, characterized in that, in a monkey challenge test, rhesus monkeys are respectively On days 0 and 28, ADS-MVA was inoculated twice, and both doses were 5 X 10 8 TCID50. Half of the vaccines were inoculated by muscle, and the other half were administered through the nasal cavity. After 4 weeks, each animal was passed through the nasal cavity with 10 5 TCID50. Vaccinated with the virus SARS-CoV PUMC01.

The invention also relates to a method for neutralization test of a pseudovirus, which is characterized in that it is carried separately The S gene of SA S-CoV and the two major plasmids of pcDNASopt9 and pNL4-3Luc+Env-Vpr- of the major core gene of Ηΐν-1 were co-transfected into 293T cells to prepare pseudovirus; the activity of neutralizing antibody was detected by pseudovirus invasion assay. Serum samples diluted in a certain proportion and the same amount of pseudovirus were cultured at 37 ° C, and serum/viral mixtures were separately added to 293-ACE2 cells, and infected cells were lysed to detect luciferase activity.

 Considering that the transferred S gene may alter the cell tropism of ADS-MVA, this may affect the safe range of the vaccine. We tested the growth characteristics of ADS-MVA in mammals. We used ADS-MVA to infect several mammalian cell lines, 293, Hela and Vera cells. Compared to the typical extended growth pattern observed in CEF cells (Fig. 2A), ADS-MVA-infected mammalian cells are confined to individual cells. Thus, although the S protein is still expressed, unlike wild-type MVA, ADS-MVA does not replicate in mammalian cells. In addition, ADS-MVA was passaged 9 times in CEF cells, but these passages did not result in the loss of S protein expression, suggesting that ADS-MVA has good genetic stability.

In the development of vaccines, recombinant vaccinia-SARS vaccine has many advantages, and it may become an ideal vaccine. The present invention utilizes modified vaccinia (MVA) as a vector to construct a SARS vaccine. Unlike traditional vaccinia, it can cause hospital infections and death. Because MVA does not contain host genes in the viral genome, it cannot replicate in humans and most mammalian cells, and MVA DNA gene expression is not affected, and early and late genes can be synthesized in human cells. More importantly, MVA has applied hundreds of millions of applications to smallpox for large-scale vaccine trials and clinical trials, and found no side effects during use, even in high-risk patients or experimental immunodeficiency monkeys. In the present invention, the full-length SARS-CoV envelope S glycoprotein gene is transferred into the MVA deletion III region gene, and we select the S protein because it acts as a major target protein for cellular and humoral immunity, mediating virus invasion, and newly produced Recombinant ADS-MVA is capable of eliciting high levels of neutralizing antibodies after two immunizations in mice, rabbits and monkeys, and neutralizing antibodies induced in rhesus monkeys prevent SARS virus invasion. The vaccine immunogenicity, vaccine safety determination, vaccine effectiveness determination, and antibody determination, viral load dynamic detection in vivo, SARS pathological changes and other indicators determine that recombinant vaccinia SARS vaccine has good immunogenicity, safety and Effectiveness, as a potentially safe and useful preventive vaccine against human and animal SARS virus infections, is safe for screening SARS for the ideal vaccine, and should be tested as soon as possible. DRAWINGS

 Fig. 1 shows an example of a method for constructing a SARS vaccine ADS-MVA.

 Figure 2 shows that S protein and GFP (green fluorescent protein) are co-expressed in ADS-MVA-infected CEF cells, wherein A shows S protein and B shows GFP.

 Figure 3 shows the construction and expression of a DNA vaccine containing an optimized S gene fragment.

 Figure 4 shows the results of detection of anti-SARS-CoV-specific neutralizing antibody responses after ADS-MVA immunization of BALB/c mice.

 Figure 5 shows the results of detection of anti-SARS-CoV-specific neutralizing antibodies in ADS-MVA immunized New Zealand white rabbits.

 Figure 6 shows the results of detection of anti-SARS-CoV-specific neutralizing antibody responses after ADS-MVA immunization of rhesus monkeys.

 • Figure 7 shows the results of anti-SARS-CoV-specific neutralizing antibody responses following immunization of New Zealand white rabbits with DNA vaccine.

 Figure 8 shows anti-SARS-CoV specific neutralization of New Zealand white rabbits (A), BALB/c mice (B), and rhesus monkeys (C) in the ACE2 receptor binding region (RBR-Fc) fused with human Fc. Adsorption and removal of antibodies.

detailed description

 Example 1

Construction of pZC3d insertion vector: The pH5 promoter via DNA synthesis was introduced into the pLW7 vector (Development of a replication-deficient recombinant vaccinia virus vaccine effective against parainfluenza virus 3 infection in an animal model. Linda) S. Wyatt. et. al. Vaccine. Vol. 14, No. 15, 1451-1456. 1996), A new generation of pZC3d vector containing a double promoter was constructed. In this vector, the S gene is under the strong synthetic promoter pSYN, the reporter gene GFP gene is transferred to the same insert in the weak promoter pH5 (Fig. 1), and GFP is used as a proxy marker for carrying the S gene. Recombinant MVA vector (provided by Dr. Bernie Moss and Dr. Lynda Wyatt of NIH, refer to Vaccine Protocols, Second Edition, August 2003, pps. 51-68, ISBN: 1-59259-399-2 Series: Methods in Molecular Medicine; Volume #: 87; by Caroline Staib and Gerd Sutter) Both Q pSYN and pH5 promoters are vaccinia-specific Promoter, a new vector allows multiple genes to be inserted into a single recombinant MVA virus. Like pLW7, the insertion site of pZC3d is the deletion region III of the MVA gene (see Figure 1).

 Example 2

 Construction and purification of ADS-MVA: The GFP gene and the S gene were constructed into pZC3d using a blunt-end ligation method. The S gene was derived from the cDNA of the SARS-CoV HKU39849 virus strain, which was isolated from Hong Kong (S gene deposited in GenBank, accession number AY278491). Recombinant ADS-MVA was produced in chicken embryo fibroblasts (CEF) by homologous recombination: First, CEF cells were infected with parental MVA (1ΜΟΙ). After 90 minutes, the same population of cells was transfected with pZC3d using Effectene (Qiagen Cat: 301427); after 48 hours, the positive cell population was selected by fluorescence emitted by GFP under a fluorescence microscope; recombinant ADS-MVA infected CEF by 8 times Cell passage was purified. Thus, using pZC3d, we recombined the wild-type SARS full-length S gene and GFP into the MVAIII region to obtain ADS-MVA (see Figure 1). For comparison, in the same manner, we introduced another modified viral ADC-MVA by introducing the modified HCV E1E2 gene into the same site.

 Example 3

Immunofluorescence assay: In order to detect S-glycoprotein expressed on the cell surface, we performed an immunofluorescence assay. A simple procedure was to place CEF cells on a 6-well plate on day 1 ( 2 x 106 cells per well), and the plates were pre-treated with Con A (10 (^g/ml) for 30 minutes and washed twice with PBS. Days, CEF cells were infected with recombinant serial dilutions of ADS-MVA (1:10). Two hours later, the cell culture medium was replaced with 2% fetal bovine serum (FCS) DMEM at 37 ° C 5 ° / Incubate for 48 hours at C0 2, and incubate the infected cells with heat-inactivated SARS patient serum (WH, 1:500) for 1 hour, then use Alexa Fluor 594-labeled goat anti-human IgG (H+L) (Molecular Probes, USA) Incubation for 30 minutes, cells were washed three times with PBS, positive colonies were identified by immunofluorescence microscopy, and the results are shown in Figure 2A. CEF cell infection control group ADC-MVA was also stained as a control in each test.

 Example 4

 Preparation of ADS-MVA strain: This strain was purified using CEF cells as described above. Purified ADS-MVA was prepared by amplification in CEF cells using a 2000 ml spinner flask culture technique.

 Example 5

Western blot analysis: in order to determine the neutralizing antibody contained in the full-length S glycoprotein in ADS-MVA In the region of the epitope, we prepared a batch of DNA vaccines containing S genes of different lengths. In order to better express the S protein in mammalian cells, we have genetically engineered the codon sequence of the S gene. The leader sequence of the S gene is replaced with a leader sequence of the tissue plasminogen activator (Tpa), which promotes protein expression by facilitating the transfer of proteins from the endoplasmic reticulum to Golgi. These DNA vaccines have been shown to be expressed in mammalian cells in transient expression assays. The main method was to transfect 293T cells with the DNA vaccine mammalian expression vector pcDNA-S200, pcDNA-S400, pcDNA-S600, pcDNA-S800, pcDNA-Sopt9, or pcDNA3.1. After 48 hours, the transfected cells (lxlO 6 ) were washed with PBS and placed in dry ice with 200 μl of cell lysate (50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 0.5% NP-40, 10% glycerol, and ^g/ml of each of pepstatin, leupeptin and pefabloc) were lysed for 30 minutes. The cell debris was centrifuged at 14,000 rpm for 10 minutes at 4 ° C, and the remaining cell lysis supernatant was electrophoresed on a 10% SDS polyacrylamide gel. The isolated protein was transferred to a nitrocellulose membrane for antibody imprinting. The results are shown in Figure 3. The S protein also binds to the N-terminal 400 amino acid rabbit antibody, and the S protein appears in the molecular bands of 160 and 250 Kd. This is larger than the actual predicted value, which may be due to post-translational processing of the protein. Positive serum samples were obtained from pcDNA-S400 sera obtained by immunizing rabbits by in vivo electroporation techniques. pcDNA-S400 encodes the N-terminal 400 amino acids of the S gene, and the negative sera are from healthy animals injected with placebo. In order to better express the S protein in mammalian cells, we have genetically engineered the codon sequence of the s gene. The leader sequence of the S gene is replaced with a leader sequence of tissue plasminogen activator (Tpa), which promotes protein expression by facilitating the transfer of proteins from the endoplasmic reticulum to Golgi. We showed that these proteins can be expressed in human 293T cells by Western blot. To ensure antibody response, we transferred the DNA plasmid into rabbits by in vivo electroporation.

 Example 6

The virus inoculation method SARS coronavirus (SARS-CoV) was isolated from SARS patients in China. The patient was a patient in the SARS ward of Peking Union Medical College Hospital. The isolated virus was cultured and passaged on Vero cells by the Union Hospital. The results were determined by RT-PCR and electron microscopy. SARS coronavirus, virus TCID50 is 10 6 /ml. The virus was named PUMC-01 strain, and its sequence is different from that of Beijing 4 strains (BJ01-4), and is closer to the SARS-CoV strain of Hong Kong SU10 strain, Singapore and Canada. Evolutionary relationship (Genbank Accession No., AY350750). The SARS coronavirus was inoculated intranasally, and all animal experiments were performed in the P3 animal laboratory according to the experimental animal use and experimental procedures of the relevant experimental animals.

 Example Ί

Immunized animals: Female BALB/c mice aged 6-8 months were immunized intramuscularly (im) with ADS-MVA at 0 and 3 weeks, and divided into the following groups according to the different immunization doses: ADS-MVA 5xl0 6 TCID50 or 5x10 7 TCID50, ADC-MVA, saline control group, a second injection after two weeks, the mice were euthanized and blood samples were analyzed. On days 0 and 28, 2 rabbits were immunized with lxl0 8 TCID50 ADS-MVA and the other two were immunized with lxl0 8 TCID50 ADC-MVA. Eight rhesus monkeys were immunized twice on days 0 and 28, of which 4 were inoculated with ADS-MVA and the other 4 were inoculated with ADC-MVA. The initial immunization dose was lxl0 8 TCID50 and the secondary immunization dose was 3x10 8 TCID50. Blood samples were taken two weeks after each immunization. DNA vaccination, in each group 400μ β DNA plasmid (pcDNA-S200, -S400, -S600 and -S800) 2 rabbits using an in vivo electroporation at 0 and 28 days, inoculated simple. Moreover, 2 months after the last DNA immunization, these rabbits were boosted every 4 weeks with lxlO 8 TCID50 ADS-MVA. Control rabbits were inoculated with a similar DNA plasmid expressing HCV E1E2, and the control group was also boosted in the same manner with ADC-MVA. In each group, serum samples were taken and analyzed 2 weeks after immunization.

In the monkey challenge test, a total of 16 3-year-old rhesus monkeys were used for the experiment, and the vaccine immunization and control groups were divided into two groups, 8 in each group, and 8 monkeys in the vaccine immunization group were numbered 0411, 0412, 0413, 0414, 0415. , 0416, 0417 and 0418; The control group 8 numbers are 0419, 0420, 0421, 0422, 0423, 0424, 0425 and 0426. The rhesus monkey was provided by the Institute of Medical Biology of the Chinese Academy of Medical Sciences. Before the inoculation, the test was carried out according to the national standard microbial SPF level of the monkey. The test showed that the SARS virus antibody was negative. Sixteen rhesus monkeys were inoculated twice on days 0 and 28, of which 8 were inoculated with ADS-MVA and the other 8 were inoculated with ADC-MVA. Both doses were 5x10 8 TCID50. Half of the vaccines were administered by intramuscular (im) vaccination and the other half were administered intranasally. After 4 weeks, each animal was inoculated intranasally with 10 5 TCID50 SARS-CoV PUMC01. It should be noted that the vaccine SARS-CoV HKU39849 virus strain and the SAS-CoV PUMC01 S gene sequence (AY350750) are completely identical. The experiment was performed in the P3 laboratory animal facility of the Institute of Laboratory Animals, Chinese Academy of Medical Sciences. We performed a neutralization test to detect the humoral immunity activity of the vaccine. The advantage of this test is to eliminate the adverse effects of using live SARS-CoV in traditional neutralization tests. We tested the serum in ADS-MVA immunized animals. Neutralizing activity produced. The activity of serum neutralizing antibodies to heat-killed live animals was tested by a pseudovirus invasion assay, which was performed according to the working methods described in the literature (refer to Chen. Z" P. Zhou, et. al. Genetically divergent strains of simian immunodeficiency virus use CCR5 as a coreceptor for entry. J. Virol. 71: 2705-14, 1997.) The pseudovirus was co-transformed with two plasmids, pcDNASopt9 and pNL4-3Luc+Env_Vpf, carrying the optimized S gene and the fflV-1 major gene, respectively. After staining 293T cells, the serum samples diluted in a certain proportion and the same amount of pseudovirus were cultured at 37 ° C for 1 hour, and then the serum/virus mixture was separately added to 293-ACE2 cells. After 56 hours, the infected cells were lysed to detect fluorescence. Enzyme activity.

First, 8 mice were immunized with ADS-MVA. Mouse M260 and M262 were immunized with 5x10 6 TCID50 vaccine, and the other 6 mice, M263, M264, MM1, MM2, MM3 and MM4 were immunized with 5x10 7 TCID50, all animals were inoculated by intramuscular injection, and the two immunizations were separated by 3 weeks. Blood samples were taken for neutralization antibody test 2 weeks after the second immunization. In the control group, 4 mice (M265, M266, M267 and M268) were inoculated with ADC-MVA expressing HCV E1E2 protein. The vaccine dose for the control group was 5x10 7 TCID50 per mouse, and we also vaccinated with normal saline. Two mice (N1 and N2) served as controls, and high levels of neutralizing antibodies were produced in 8 mice immunized with ADS-MVA. In the high-dose group, serum diluted more than 1000-fold still inhibited or neutralized 50% of the virus (IC50). Mice in the two control groups did not produce neutralizing antibodies. In addition, there was a significant dose-related relationship between the two ADS-MVA dose groups (see Figure 4), therefore, ADS-MVA induced SARS-CoV-specific neutralizing antibodies in mice.

To investigate whether similar neutralizing antibodies can be produced in other strains of animals, we immunized two rabbits with ADS-MVA. The immunization procedure differed at the second immunization, this time at 4 weeks of immunization, and the dose selection was based on the results of our previous test for MVAHIV-1 vaccine in small animals. High levels of SARS-CoV-specific neutralizing antibodies were generated in two rabbits (R524 and R525) inoculated with ADS-MVA. The IC50 was still detectable after 10,000-fold dilution of the serum. Rabbits (R520 and R521) inoculated with ADC-MVA in both control groups did not produce SARS-CoV neutralizing antibodies (see Figure 5). These data show that high levels of neutralizing antibodies produced by ADS-MVA are not It has variety specificity. All of the animals tested were well tolerated with this vaccine and no significant disease or weight loss was observed in the experiment.

 To explore the possibility of using ADS-MVA in humans, we further tested ADS-MVA in rhesus monkeys. A total of 16 rhesus monkeys were used in this experiment, 8 were immunized with ADS-MVA, and 8 were immunized with ADC-MVA, and serum samples were continuously taken to detect the presence of neutralizing antibodies. ADS-MVA-immunized rhesus monkeys produced high levels of SARS-CoV neutralizing antibodies compared to the control group ADC-MVA. Therefore, in addition to small animals, ADS-MVA can also produce neutralizing antibodies in rhesus monkeys. Considering that SARS-CoV mainly infects the respiratory system, we also tested the titer of neutralizing antibodies in the lungs and bronchi, lung Simultaneously taken with bronchial tissue samples and serum, but no neutralizing antibodies were detected in all 8 rhesus monkeys, even at the lowest dilution (<1:10) (see Figure 6).

 In addition, rabbits were immunized with DNA vaccines S200 (R416, R417), S400 (R418, R419), S600 (R683, R684) and DNA vaccines expressing HCVE1E2 (R518, R519) using in vivo electroporation techniques. Same as above, all were immunized twice, at intervals of 4 weeks, and the results are shown in Figure 7.

 Example 9

Clinical observation and blood test: The body temperature of the 16 animals before the experiment was (37.8~38.rC), and no increase in body temperature was observed after vaccination and control solution. 16 rhesus monkeys had an increase in body temperature on days 1 to 5 of virus inoculation, ranging from 38.6 to 39.2 °C. Some control animals had difficulty breathing, reduced feeding, and reduced weight. No clinical manifestations such as cough, runny, vomiting, or diarrhea. The average number of leukocytes in the first 8 vaccinated monkeys was (7.6±3.1) X 10 3 , and there was no significant change after vaccination and virus vaccination; 8 control monkeys averaged (6.9±2.9)×10 3 before infection, virus inoculation After 2, 5, 7 and 14 days, the mean range was (3.9 ± 1.8 to 5.4 ± 1.9) X 10 3 . In blood biochemical assays, AST increased significantly at various time points after control monkey immunization with virus, but there was a significant increase at 7 and 14 days after vaccination with vaccinated monkey virus. Other indicators such as ALT, ALP, UREA, CRE, LDH, CK, TP There was no significant change in ALB and throughout the experiment.

 Example 10

Antibody adsorption assay: A soluble recombinant ACE2 receptor binding region (S310-510) fused to human Fc (RBR-Fc) was used to adsorb antibodies that specifically bind to the receptor binding region. The RBR-Fc/antibody complex was then removed with specific anti-human IgG Fc-agarose (Sigma). Diluted serum and soluble The RBR-Fc was incubated for 45 minutes at room temperature and then pre-washed anti-human IgG Fc agarose was added to each well. The microplate was shaken at room temperature for about 2 hours to prevent gel precipitation and adsorption. The microplates were centrifuged at 3000 rpm for 10 minutes at room temperature, and the remaining unbound serum was neutralized using a pseudovirus. To ensure the specificity of the assay, parallel controls were placed in the microplates, including the anti-human IgG gel and the RBR-Fc two groups. In another set of experiments, since the monkey serum containing the antibody cross-reacted with the anti-human Fc antibody coated on the carrier gel beads, we had to take a different approach, we used one of the chemicals described in the product manual. Methods Sepharose 4 Fast Flow beads (Amersham Biosciences) were directly bound to RBR-Fc. Instead of adsorbing the antibody with soluble RBR-Fc, the RBR-Fc binding beads were used to adsorb and remove the monkey antibody that binds to the RBR portion, and the rest of the experimental methods were the same as before.

 The sera of rabbits and mice were diluted 1:300 to 1:600 times according to the results of the neutralization antibody test in order to eliminate the influence of excessive neutralizing antibodies on the experimental results. Soluble RBR-Fc can adsorb and remove rabbit immune serum. Most of the neutralizing antibodies in the RBR-Fc (-) group had no effect on neutralizing antibodies in the agarose beads. Interestingly, the neutralizing antibody activity in the serum of the DNA vaccine-immunized animals (R681.D and R682.D) was almost completely eliminated. Correspondingly, the animals immunized with the ADS-MVA vaccine retained some of the neutralizing activity in the serum after adsorption, suggesting that ADS-MVA can induce a broad spectrum of neutralizing antibodies. ADS-MVA produces a broader spectrum of neutralizing antibody activity than the S600 DNA vaccine, ie, the full-length S protein may have multiple neutralizing antibody active sites (see Figure 8A).

 Mice were immunized with ADS-MVA and the same results were found. The vast majority of mouse neutralizing antibodies are adsorbed or removed by RBR-Fc recombinant proteins, so our data show a major neutralizing antibody-determining site in the ACE2 binding region (see Figure 8B).

In the test monkey serum test, RBR-Fc-conjugated Sepharose 4 Fast Flow beads were used to adsorb and remove neutralizing antibodies bound to the RBR fraction in monkey serum. As shown in Figure 8C, beads without RBR-Fc (-) had no effect on the activity of neutralizing antibodies; whereas RBR-Fc beads (+) were able to remove most neutralizing antibodies, a result similar to rabbits and mice. Serum test results. Based on our experimental results, we conclude that ADS-MVA induces neutralizing antibodies, first targeting the binding region of the SARS-CoV receptor ACE2. This conclusion is reflected in all three species, and suggests that neutralizing antibodies produced by ADS-MVA are not species-specific (see Figure 8C). SARS-CoV isolation test: After inoculation of rhesus monkeys with SARS-CoV virus, nasopharyngeal swab specimens were collected on days 2, 5, and 7 and fully immersed in lml DMEM medium, filtered through 0.22μηι filter, and inoculated with Vero. The cells were adsorbed for 1 hour and the virus was isolated. They were euthanized 7 days and 30 days after challenge, and the lung tissues of each animal were homogenized for virus isolation. The lung tissue homogenate was inoculated into Vero cells for virus isolation. After adsorption for 1 hour, the medium was replaced by fresh medium after elution. The culture was observed for 10 days after cytopathic effect (CPE). If no CPE was found, the second batch of Vera cells were blindly inoculated with the culture medium for another 10 days. If CPE is found, the SARS-CoV-specific antigen in Vero cells is detected by immunofluorescence assay (same as above) to avoid false positives. Separation test results are shown in Table 1.

 Example 12

 Reverse transcription polymerase chain reaction (RT-PCR): Viral RNA was detected in specimens of SARS-CoV virus-inoculated rhesus monkeys. Total RNA was isolated from various specimens using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Diagnostics). The isolated RA was tested using a commercial SARS-CoV RT-PCR kit (Roche Diagnostics). The results are shown in Table 1.

 Table 1. Examination of the virus after inoculation of SARS-CoV in Chinese rhesus monkeys;

Rhesus Nab titer virus isolation** RT-PCR (days) ***

 ADS-MVA

 Rh0412 2259 - - . - -

Rh0413 1523 - + - -

Rh0416 3634 - - - -

Rh0417 9531 - - - - Control MVA

 Rh0420 - - + + +

Rh0421 - + + + +

Rh0424 - - ― + +

Rh0425 - + - + +

 *Detecting neutralizing antibodies before virus inoculation

 **Separation of virus from lung tissue homogenate

*** RT-PCR of nasopharyngeal swab specimens Pathological tissue section preparation: In the animals sacrificed on the 7th day and one month after virus inoculation, the lungs and other tissues were taken, fixed in 10% formaldehyde, embedded in paraffin, and routinely pathologically sectioned, HE stained, and microscopically examined. The results showed that the vaccine-immunized group showed pulmonary interstitial inflammation in 2 animals after 7 days of challenge. After 35 days of challenge, 1 animal developed interstitial lung inflammation, and pulmonary pleural edema appeared. The other 3 animals showed focal Pulmonary interstitial inflammation; The control group showed that there were pulmonary interstitial inflammation in 4 animals after 7 days of challenge, and the degree of lesion was heavier. The size of lesions in each animal was slightly different. After 35 days of challenge, there were pulmonary interstitial inflammation in 4 animals. The degree of lesions was lighter than that of 4 animals after 7 days of challenge. The size of lesions in each animal was slightly different.

 From the above pathological comparison results, compared with the model group, the lung lesions of the vaccine group were lighter, and the preliminary test results suggested that the SARS-related vaccine tested could alleviate the SARS virus attack on the test animals rhesus monkeys; 2 The vaccine showed no pathological changes in the lesions of other organs examined.

Claims

Claim
1. A SARS-CoV vaccine ADS-MVA constructed on the basis of attenuated vaccinia virus MVA.
The SARS vaccine according to claim 1, which is an MVA vaccine in which the S gene is inserted into the modified attenuated vaccinia MVA deletion region III.
 3. A method of preparing a SARS-CoV vaccine, comprising:
 a. constructing a shuttle plasmid comprising a SARS-CoV antigen gene, and an attenuated vaccinia virus MVA deletion III region gene coding region sequence or a flanking sequence homologous to a sequence flanking the coding region; b. allowing cells in the vaccinia virus The shuttle plasmid is homologously recombined with the attenuated vaccinia virus MVA strain;
 c The single-spot screening of the recombinant virus was performed by screening markers to isolate a pure highly attenuated vaccinia ADS-MVA virus.
 The method for producing a SARS-CoV vaccine according to claim 3, wherein the shuttle plasmid is a shuttle plasmid in which a SARS-CoV antigen gene is located downstream of the promoter pSYN, and a selection marker is located downstream of the promoter pH5.
 The method of producing a SARS-CoV vaccine according to claim 4, wherein the screening marker is GFP, and the SARS-CoV and GFP genes are transferred into the same insertion sequence.
 The SARS-CoV vaccine produced by the method for producing a SARS-CoV vaccine according to any one of claims 3 to 5.
 7. The recombinant vaccine of claim 1 or 2 SARS-CoV vaccine The use of ADS-MVA in the treatment or prevention of SARS-CoV infection and disease.
 The use of the method for constructing attenuated recombinant vaccinia SARS vaccine ADS-MVA according to any one of claims 3 to 5 for constructing a recombinant MVA for expression of a foreign gene.
 The method for constructing attenuated recombinant vaccinia SARS vaccine ADS-MVA according to any one of claims 3 to 5, for use in the construction of a recombinant MVA expressing exogenous pathogen antigen for the preparation of a pathogen detecting and diagnostic reagent.
The method for preventing pathogenic SARS-CoV infection in a Chinese rhesus monkey by the attenuated recombinant vaccinia SARS-CoV vaccine ADS-MVA according to claim 1 or 2, wherein in the monkey challenge test, the constant Rhesus monkeys were inoculated with ADS-MVA twice on days 0 and 28, respectively. It was 5 X 10 8 TCID50, half of the vaccines were vaccinated by muscle, and the other half were administered intranasally. After 4 weeks, each animal was inoculated with the virus SARS-CoV by nasal infusion with 10 5 TCID50.
 A method for neutralization test of a pseudovirus according to claim 1 or 2, characterized in that pcDNASopt9 and p I^-SLuc^ carrying the S gene of SARS-CoV and the major core gene of HIV-1, respectively, are used. Env-Vpr—Two plasmids were co-transfected into 293T cells to prepare pseudoviruses; neutralizing antibody activity was detected by pseudovirus invasive assay, serum samples diluted in a certain proportion were cultured with the same amount of pseudovirus, and serum/viral mixture were separately Incorporation into 293-ACE2 cells, lytic cell lysis was performed to detect luciferase activity.
PCT/CN2006/000181 2005-01-27 2006-01-27 A recombinant sars-cov vaccine comprising attenuated vaccinia virus carriers WO2006079290A1 (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1562365A (en) * 2003-05-21 2005-01-12 中山大学肿瘤防治中心 SARS vaccine of adenovirus carrier and preparation method, application of coronavirus S gene

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1562365A (en) * 2003-05-21 2005-01-12 中山大学肿瘤防治中心 SARS vaccine of adenovirus carrier and preparation method, application of coronavirus S gene

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Title
BISH H. ET AL.: 'Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice' PROC. NATL. ACAD. SCI. USA vol. 101, no. 17, 27 April 2004, pages 6641 - 6646, XP002332532 *
CHEN Z. ET AL.: 'Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region' J. VIROL. vol. 79, no. 5, March 2005, pages 2678 - 2688 *
CZUB M. ET AL.: 'Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets' VACCINE vol. 23, no. 17-18, 18 March 2005, pages 2273 - 2279, XP004777538 *
DONG GUANMU ET AL.: 'Research advancement on SARS vaccines' CHIN. J. BIOLOGICALS vol. 17, no. 1, January 2004, pages 64 - 66 *
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