HSV attenuated strain and preparation method and application thereof
阅读说明：本技术 一种hsv减毒株及其制备方法与应用 (HSV attenuated strain and preparation method and application thereof ) 是由 李艳梅 李一濛 徐沐 陈姝妮 于丽 易庭 郑丽春 于 2021-07-28 设计创作，主要内容包括：本发明涉及一种HSV减毒株及其制备方法与应用,属于生物技术领域,本发明针对HSV1和HSV2两种病毒中均具有的、能特异性抑制天然免疫反应/获得性免疫反应/细胞应激保护性反应的不同产物,以及能够进入机体神经系统导致潜伏性感染的能力,设计了一种能够基于缺失修饰病毒不同基因而改变病毒在长期进化过程中形成的、特异性针对宿主细胞应激反应和天然免疫反应的不同信号传导途径和不同效应靶点的作用特性的技术策略,从而得到了一个特定的HSV1基因缺陷减毒毒株；本发明所得HSVI基因缺陷减毒毒株作为治疗及预防疱疹病毒HSV1的疫苗使用,能有效治疗或预防疱疹病毒HSV1的感染所引起的疾病。(The invention relates to an HSV attenuated strain and a preparation method and application thereof, belonging to the technical field of biology, aiming at different products which are contained in two viruses, namely HSV1 and HSV2 and can specifically inhibit natural immunoreaction/acquired immunoreaction/cell stress protective reaction, and the capability of entering a nervous system of an organism to cause latent infection, the invention designs a technical strategy which can change the action characteristics of different signal transduction paths and different effect targets which are formed in the long-term evolution process of the viruses and specifically aim at the host cell stress reaction and the natural immunoreaction based on deletion modification of different genes of the viruses, thereby obtaining a specific HSV1 gene defect attenuated strain; the HSVI gene defect attenuated strain obtained by the invention can be used as a vaccine for treating and preventing the herpes virus HSV1, and can effectively treat or prevent diseases caused by infection of the herpes virus HSV 1.)
1. An HSV attenuated strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.21598, and the preservation date is 2021, 07 months and 13 days.
2. The method for preparing an attenuated strain of HSV as in claim 1, wherein HSV1 is subjected to modified reprogramming, which comprises: cleaving a fragment that excludes nucleotides 185 to 329 of a Us5 gene of HSV1 virus; splicing to remove the gene of the UL41 gene from the 249 to 435 coding region; a gene which is excised from the coding region from position 91 to position 289 of the Us11 gene; the gene fragment encoding the nucleotide sequence from position 207 to 453 of the RL1 gene sequence was excised.
3. The method of preparing an attenuated strain of HSV as in claim 2, comprising the steps of:
(1) designing a primer A, shearing a gene fragment of 185 th to 329 th nucleotides of a Us5 gene coding region of the HSV virus in the presence of a Cas9 enzyme, and cloning, purifying and screening to obtain a Us5 gene partially sheared virus strain which is named as WR-1;
(2) designing a primer B on the basis of WR-1, and shearing a gene fragment of the UL41 gene from 249 to 435-bit coding region nucleotide in the presence of Cas9 enzyme to obtain a strain with the UL41 gene partially deleted, wherein the strain is named as WR-2;
(3) designing a primer C on the basis of WR-2, cutting a nucleotide gene fragment from 91 to 289 bit of a Us11 coding region in the presence of a Cas9 enzyme, and performing cloning, purification and screening to generate a WR-3 strain;
(4) on the basis of WR-3, a primer D is designed, a gene segment which codes from 207 to 453 bit nucleotide sequences in an RL1 gene sequence is removed in the presence of a Cas9 enzyme, and an HSV1 attenuated strain is obtained through cloning, purifying and screening.
4. The attenuated strain of HSV1 of claim 3, further comprising:
the nucleotide sequence table of the primer A is as follows: US5-F1: CACC GGGCTTTTGGGAAGCCTCGT; US5-R1: AAAC ACGAGGCTTC CCAAAAGCCC; US5-F2: CACC GGGCTGTGCGCCGTAGTCCT; US5-R2: AAAC AGGACTACGG CGCACAGCCC;
the sequence of the cut Us5 gene is shown as SEQ ID NO:1
The nucleotide sequence table of the primer B is UL41-F1: CACC CATCTTCGTTACCGATCGCG; UL41-R1: AAAC CGCGATCGGT AACGAAGATG; UL41-F2: CACC CGCGGCACCTCTCTGG CCTC; UL41-R2: AAAC GAGGCCAGAG AGGTGCCGCG;
the sequence of the sheared UL41 gene is shown as SEQ ID NO. 2;
the nucleotide sequence table of the primer C is US11-F1: CACC TCCGCCG GCATGCACCC CAG; US11-R1: AAAC CTGGGGTGCA TGCCGGCGGA; US11-F2: CACC GCGAGCTCCC AGAGACCCCA; US11-R2: AAAC TGGGGTCTCT GGGAGCTCGC;
the sequence of the cut Us11 gene is shown as SEQ ID NO. 3;
the nucleotide sequence table of the primer D is RL1-F1: CACC GAGTCCGCGT CCGACGACGA; RL1-R1: AAAC TCGTCGTCGG ACGCGGACTC; RL1-F2: CACC CGCCTGCG CCTGCGACGC GC; RL1-R2: AAAC GCGCGTCGCA GGCGCAGGCG;
the sequence of the sheared RL1 gene is shown as SEQ ID NO. 4.
5. The attenuated strain of HSV according to claim 3 or 4, for its preparation, characterized in that: in the steps (1) to (4), the cloning, purifying and screening method comprises the following steps:
spreading Vero cells in a 6-well plate until the cells are completely attached to the wall, discarding the original culture medium, washing with PBS, and culturing the mutant virus in serum-free MEM (minimum essential medium) at a ratio of 1:1-1:106After dilution, virus solutions with different dilutions are added into each cell well according to the volume of 1ml per well, and 1 negative control well is arranged;
placing in 5% CO2Adsorbing for 2-3h in an incubator at 37 ℃;
then, after the virus liquid in the cell culture plate is discarded, slowly adding the agar culture medium mixture which is subjected to high pressure and is cooled to about 42 ℃ into the cell pores along the plate wall according to the volume of 2.5-3 ml/pore;
cooling, and adding 5% CO2Culturing at 37 deg.C in inverted state, and observing for 3-4 days until plaque appears; after the macroscopic plaques appeared, the plaques were picked after labeling and resuspended in 100ul serum-free DMEM medium.
6. The attenuated strain of HSV according to claim 3 or 4, for its preparation, characterized in that: in the steps (1) to (4), the propagation of the virus strain is carried out before the cloning, purification and screening.
7. Use of an attenuated strain of HSV according to claim 1 in the preparation of a therapeutic or prophylactic herpes vaccine.
8. A genetically modified attenuated vaccine of the herpes virus HSV1, comprising the attenuated strain of HSV of claim 1.
9. Use of an attenuated strain of HSV according to claim 1 in the manufacture of a vaccine for the treatment or prevention of a disease associated with the infection of the herpes virus HSV 1.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an HSV attenuated strain as well as a preparation method and application thereof.
Background
Human Herpes simplex virus includes two types, HSV1 and HSV2(Herpes simplex virus 1, 2). Both belonging to members of the alpha subgroup of the human herpesvirus family. Both viruses cause herpetic diseases of various ages in humans, including oral herpes, genital herpes, herpes encephalitis, and other infections of the herpetic nervous system. Among them, HSV1 is commonly found in oral herpes, and HSV2 is commonly found in genital herpes. However, the infection patterns of both tend to be interconvertible. Both HSV1 and HSV2 have a high rate of infection and morbidity worldwide. Different statistics indicate that the infection rate of HSV1 in infected people is about 25-47%, and HSV2 in people between 15 and 49 years old is about 11-15%. Among them, the rate of infection of neonatal herpes encephalitis by genital infection is about 0.1%, which leads to higher mortality. To date, no effective therapeutic drugs and vaccines against these two viruses have been developed. Therefore, the basic and technical researches related to the vaccine aiming at the virus have important significance.
Disclosure of Invention
In order to overcome the problems in the background technology, the invention provides an HSV attenuated strain and a preparation method and application thereof, and HSV1 is modified and reprogrammed by using a gene editing technology, so that the HSV1 becomes a vaccine candidate strain with immune effectiveness and safety prospects. Therefore, one of the purposes of the invention is to provide an HSV attenuated strain; the second purpose of the invention is to provide a preparation method of HSV attenuated strain; the invention also aims to provide a vaccine containing the HSV attenuated strain, and the fourth aim of the invention is to provide the application of the HSV attenuated strain as the vaccine.
The first purpose of the invention is realized by the following technical scheme:
the HSV attenuated strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is classified and named as: human herpes simplex virus 1 (Herpse simplex virus 1) with the preservation number of CGMCC No.21598 and the preservation unit: china general microbiological culture Collection center, preservation Address: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, and the preservation date is 2021 year, 07 month and 13 days.
The second purpose of the invention is realized by the following technical scheme:
the preparation method of the HSV attenuated strain is realized by modifying and reprogramming HSV1, and specifically comprises the following steps: cleaving a fragment of nucleotide 185 to 329 of a Us5 gene of an HSV virus; splicing genes of the UL41 gene from 249 to 435 coding regions; cleaving a nucleotide gene fragment from position 91 to position 289 of the coding region of the Us11 gene; the gene fragment encoding the amino acid sequence from 207 to 453 of the RL1 gene sequence was excised.
Further, the method comprises the following steps:
(1) designing a primer A, shearing a gene fragment of 185 th to 329 th nucleotides of a Us5 gene of the HSV virus in the presence of a Cas9 enzyme, and cloning, purifying and screening to obtain a Us5 gene partially sheared virus strain which is named as WR-1;
(2) designing a primer B on the basis of WR-1, shearing a gene fragment of a coding region from 249 to 435 of the UL41 gene in the presence of a Cas9 enzyme, and carrying out cloning, purification and screening to obtain a strain with a partially deleted UL41 gene, wherein the strain is named as WR-2;
(3) designing a primer C on the basis of WR-2, cutting a nucleotide gene fragment from 91 to 289 th position of a coding region of a Us11 gene in the presence of a Cas9 enzyme, and performing cloning, purification and screening to generate a WR-3 strain;
(4) on the basis of WR-3, a primer D is designed, a gene segment from 207 to 453 nucleotides in the coding region of an RL1 gene is removed in the presence of a Cas9 enzyme, and an HSV1 attenuated strain is obtained through cloning, purifying and screening.
Further, in the above-mentioned case,
the nucleotide sequence of a primer A used for cutting the Us5 gene is US5-F1: CACC GGGCTTTTGGGAAGCCTCGT; US5-R1: AAAC ACGAGGCTTC CCAAAAGCCC; US5-F2: CACC GGGCTGTGCGCCGTAGTCCT; US5-R2: AAAC AGGACTACGG CGCACAGCCC.
The gene sequence of the cut Us5 gene is shown as SEQ ID NO. 1.
The nucleotide sequence table of a primer B used for cutting the UL41 gene is UL41-F1: CACC CATCTTCGTTACCGATCGCG; UL41-R1: AAAC CGCGATCGGT AACGAAGATG; UL41-F2: CACC CGCGGCACCTCTCTGG CCTC; UL41-R2: AAAC GAGGCCAGAG AGGTGCCGCG.
The sequence of the sheared UL41 gene is shown as SEQ ID NO. 2.
The nucleotide sequence table of a primer C used for shearing a Us11 gene is US11-F1: CACC TCCGCCG GCATGCACCC CAG; US11-R1: AAAC CTGGGGTGCA TGCCGGCGGA; US11-F2: CACC GCGAGCTCCC AGAGACCCCA; US11-R2: AAAC TGGGGTCTCT GGGAGCTCGC.
The sequence of the cut Us11 gene is shown as SEQ ID NO. 3.
The nucleotide sequence table of a primer D used for shearing RL1 gene is RL1-F1: CACC GAGTCCGCGT CCGACGACGA; RL1-R1: AAAC TCGTCGTCGG ACGCGGACTC; RL1-F2: CACC CGCCTGCG CCTGCGACGC GC; RL1-R2: AAAC GCGCGTCGCA GGCGCAGGCG.
The sequence of the sheared RL1 gene is shown as SEQ ID NO. 4.
Further, in the steps (1) to (4), the clone purification screening method comprises the following steps:
spreading Vero cells in a 6-well plate until the cells are completely attached to the wall, discarding the original culture medium, washing with PBS, and diluting the mutant virus 10 times with serum-free MEM (1:1-1: 10)6) Different dilutions of virus fluid were then added to each cell well at a volume of 1ml per well and 1 negative control well was set. Placing in 5% CO2Adsorbing at 37 deg.C for 2-3 hr. Subsequently, the fines are discardedAfter the virus solution in the cell culture plate is slowly added to the cell wells along the plate wall in a volume of 2.5-3 ml/well, an agarose medium mixture which has been autoclaved and allowed to cool to about 42 ℃. Cooling, and adding 5% CO2Culturing at 37 deg.C in inverted state, and observing for 3-4 days until plaque appears;
until visible plaques appeared, and after labeling, the plaques were picked and resuspended in 100ul serum-free DMEM medium.
Further, in steps (1) to (4), cell propagation is performed before clone purification screening.
The invention provides an application of an HSV attenuated strain in preparation of herpes vaccines.
A herpes vaccine comprising an attenuated strain of HSV of the present invention.
The invention has the beneficial effects and the technical principle that:
aiming at different products which are contained in two viruses, namely HSV1 and HSV2 and can specifically inhibit natural immune response/acquired immune response/cell stress protective response and enter the nervous system of an organism to cause latent infection, the research designs a technical strategy which can change the action characteristics of different signal conduction paths and different effect targets which are formed in the long-term evolution process of the viruses and are specifically aimed at host cell stress response and natural immune response based on deletion modification of different genes of the viruses, combines a series of a plurality of coding genes which can systematically control the transcriptional activation of the viruses when the viruses inhibit the cell response step by step based on the pathological mechanism of the host cell infection process of the HSV, and inactivates the virus genes which can generate pathological invasion to the cell physiological stress and the natural immunological response function, thus, a specific HSV1 gene-deficient attenuated strain is obtained.
Drawings
FIG. 1 is an electrophoretogram showing the PCR identification result of WR-1 virus;
FIG. 2 is an electrophoretogram of the PCR identification result of WR-2 virus;
FIG. 3 is an electrophoretogram showing the PCR identification result of WR-3 virus;
FIG. 4 is an electrophoretogram of the PCR identification result of WR-4 virus;
FIG. 5 is a graph showing the proliferation kinetics of WR-4 and wild strains in diploid epithelial cells;
FIG. 6 is a graph showing the proliferation kinetics of WR-4 and wild strain in SH-SY5Y cells;
FIG. 7 is a plaque morphology on vero cells of WR-4 and the wild strain;
FIG. 8 is a graph showing the weight change of WR-4 strain and wild strain in mice infected with nasal drops;
FIG. 9 is a graph showing the survival rate of WR-4 strain and wild strain infected mice by nasal drip;
FIG. 10 difference in brain viral load between strain WR-4 and the wild type strain;
FIG. 11 difference in spinal cord viral load between strain WR-4 and wild type strains;
FIG. 12 difference in viral load in trigeminal ganglia between strain WR-4 and wild strain;
FIG. 13 changes in TNF- α expression in serum following infection of mice with WR-4 strain and wild-type strain;
FIG. 14 changes in IFN- β expression in serum following infection of mice with WR-4 strain and wild-type strain;
FIG. 15 IFN-. gamma.expression changes in serum after infection of mice with WR-4 strain and wild-type strain;
FIG. 16 weight change in mice following treatment of strain WR-4 with respect to HSV1 infection;
FIG. 17 change in survival of mice following treatment of strain WR-4 with respect to HSV1 infection;
FIG. 18WR-4 strain changes in viral proliferation in brain tissue following treatment for HSV1 infection;
FIG. 19WR-4 Strain changes in viral proliferation in spinal cord tissue following treatment for HSV1 infection;
FIG. 20WR-4 Strain changes in viral proliferation in trigeminal ganglion tissues following treatment for HSV1 infection;
FIG. 21 changes in IFN-. gamma.expression in serum after treatment of strain WR-4 against HSV1 infection;
FIG. 22WR-4 Strain CD8+ T subset cell changes in peripheral blood following treatment for HSV1 infection.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
Example 1 attenuated strains of HSV and methods for their preparation
The embodiment provides an HSV attenuated strain which is preserved in the center of common microorganisms of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.21598, and the preservation date is 2021, 07 months and 13 days. The HSV attenuated strain is obtained by modifying and shearing a Us5 gene, an UL41 gene, a Us11 gene and an RL1 gene of HSV 1.
Except for modifying a Us5 gene, a UL41 gene, a Us11 gene and a RL1 gene, the gene sequences of the HSV attenuated strain are the same as those of an HSV1 gene.
The sequence of the modified Us5 gene is shown in SEQ ID NO 1.
The sequence of the modified UL41 gene is shown in SEQ ID NO. 2.
The sequence of the modified Us11 gene is shown in SEQ ID NO 3.
The modified RL1 gene sequence is shown in SEQ ID NO. 4.
The preparation method of the HSV attenuated strain comprises the following steps:
(1) designing a primer A: US5-F1: CACC GGGCTTTTGGGAAGCCTCGT; US5-R1: AAAC ACGAGGCTTC CCAAAAGCCC; US5-F2: CACC GGGCTGTGCGCCGTAGTCCT; US5-R2: AAAC AGGACTACGG CGCACAGCCC.
After the primer transfects 293 cells infected by HSV 1F strains, the fragments of 185 th to 329 th nucleotides of the Us5 gene of HSV1 virus can be cut in the presence of Cas9 enzyme, the fragments are continuously propagated in the cells for 48 hours after being cut and recombined, and the virus strain with the Us5 gene partially cut is obtained after cloning, purifying and screening, and is named as WR-1. The sequence of the cut Us5 gene is shown as SEQ ID NO: 1.
When a virus infects a host cell, the stimulation to the cell can cause cell physiological stress response, wherein one main content is cell apoptosis, and the physiological response can lead the host cell which is proliferated by the virus to rapidly die by itself, thereby inhibiting the proliferation of the virus to a certain extent. The encoded product of the Us5 gene of HSV virus can act on apoptosis-related protein and play a role of kinase, the apoptosis initiation process is stopped on the basis of changing the apparent biological property of the protein, and the pathological damage of the virus to host cells is reduced by deleting the related coding region sequence of the virus Us5 gene.
(2) On the basis of WR-1, a primer B is designed: UL41-F1: CACC CATCTTCGTTACCGATCGCG; UL41-R1: AAAC CGCGATCGGT AACGAAGATG; UL41-F2: CACC CGCGGCACCTCTCTGG CCTC; UL41-R2: AAAC GAGGCCAGAG AGGTGCCGCG. After the primer transfects 293 cells infected by WR-1 strains, genes of the UL41 gene from 249 to 435 coding regions can be cut in the presence of Cas9 enzyme, the genes are continuously propagated in the cells for 48 hours after cutting recombination, and the strains with UL41 gene partially deleted are obtained through cloning, purifying and screening, and are named as WR-2. The sequence of the sheared UL41 gene is shown as SEQ ID NO. 2.
During the process of infecting cells, HSV can block the cell stress response-cell autonomous programmed death by utilizing the gene product of the coded Us5, and can also increase the pathological hijacking of the cells through a multifunctional protein. The protein is encoded by UL41, namely Vhs protein (Virion host shutoff protein), which can selectively degrade the mRNA of host cells by utilizing the specific endonuclease activity of the mRNA, and the process has an important effect on another important loop of the cells in the stress reaction process, namely, activation of an interferon system, encoding of various specific viral factors to limit the diffusion of viruses among cells, so that the cells directly destroy the reaction process of the infected cells to send early warning signals to surrounding uninfected cells and generate antiviral effector molecules while destroying the virus infected cells by a programmed death (apoptosis) mechanism to limit the proliferation of the viruses. Meanwhile, because Vhs also has the function of acting on an RLRs receptor (i.e. a receptor of a polypeptide-acid-indicator gene 1 like) in a natural immune response system in cells, since such receptors are distributed on the surface of organelles, they are able to recognize heterologous DNA molecules that bind invading pathogens, simultaneously activates a relevant cGAS/STING mediated DNA molecule recognition channel, thereby activating the NF-kB transcription system of cells to generate a series of corresponding natural immune response signal molecules, it is used as the first-stage immune system in epithelial tissue of body, and can activate natural immunoreaction cells such as dendritic cell, NK cell and macrophage, etc., so that it can chemotaxis infected cell site, phagocytose infected cell or pathogenic molecule, and can be used as antigen to deliver it to T cell in acquired immune system to activate specific antiviral reaction. Therefore, when the virus infects epithelial cells, the normal activation of the normal innate-adaptive immune response process of the body can be directly influenced by the fact that Vhs molecules act on the receptors of RLRs and block the stimulation of the innate immune response. This therefore results in HSV infection, every link in the host cell stress response-innate immune response-adaptive immune response is affected. By destroying the inhibitory molecule Vhs coding gene UL41 of the virus to the specific mRNA molecule in the cell stress response, the interference effect of the virus to the RLRs in the cells is controlled, and the smooth activation of the natural immune response in the cells is ensured.
(3) On the basis of WR-2, a primer C is designed: US11-F1: CACC TCCGCCG GCATGCACCC CAG; US11-R1: AAAC CTGGGGTGCA TGCCGGCGGA; US11-F2: CACC GCGAGCTCCC AGAGACCCCA; US11-R2: AAAC TGGGGTCTCT GGGAGCTCGC. After the primer transfects 293 cells infected by WR-2 strains, nucleotide gene fragments from 91 to 289 bits of the coding region of the Us11 gene can be cut in the presence of Cas9 enzyme, the DNA fragments are continuously propagated in the cells for 48 hours after cutting and recombination, and the WR-3 strains are obtained after cloning, purifying and screening. The sequence of the cut Us11 gene is shown as SEQ ID NO. 3.
Under the premise that a WR-2 strain can remove the genetic basis of virus inhibition cell stress response/natural immune response, corresponding gene modification mutation is carried out on a protein coded by a Us11 gene which has the function of blocking the activation of host cell PKR enzyme so as to inhibit the synthesis of cell protein, so that the inhibition effect of the virus on the cell response is reduced, the capability of inhibiting eIF 2-alpha subunit phosphorylation of ICP34.5 protein coded by RL1 gene of the virus is also realized according to the capability of assisting the phosphorylation of the eIF 2-alpha subunit of the Us11 coded protein, and the measure can simultaneously reduce the function of ICP34.5 when the action function of the Us11 coded protein on the PKR is removed, and has stronger attenuation effect on the comprehensive toxicity of the virus strain in the process of infecting a host.
(4) On the basis of WR-3, a primer D is designed: RL1-F1: CACC GAGTCCGCGT CCGACGACGA; RL1-R1: AAAC TCGTCGTCGG ACGCGGACTC; RL1-F2: CACC CGCCTGCG CCTGCGACGC GC; RL1-R2: AAAC GCGCGTCGCA GGCGCAGGCG. After the primer transfects 293 cells infected by WR-3 strains, gene segments encoding amino acid sequences from 207 to 453 sites in RL1 gene sequences can be removed in the presence of Cas9 enzyme, the cells are continuously propagated for 48 hours after shearing and recombination, and HSV attenuated strains are obtained through cloning, purifying and screening, and are named as WR-4. The sequence of the sheared RL1 gene is shown as SEQ ID NO. 4.
The inventor makes further analysis on the causes of pathological injury possibly existing for a long time in HSV infection, failure of an immune system to completely eliminate viruses and the like, and based on the characteristic that the virus can inhibit physiological stress/natural immune response/acquired immune response of an organism at different levels in the infection process and the long-term latency of the virus in nerve cells (mainly trigeminal ganglion and sacral ganglion) caused by the virus, so that the virus gets rid of the monitoring and elimination of the immune system in the process of infecting the organism and becomes a difficult problem in vaccine research, based on the analysis and reasoning, and based on the removal of three important genes of the virus which interferes with the physiological stress/natural immune response/acquired immune response of cells by means of genetic modification, namely, the Us5/UL41/Us11, the RL1 gene (which encodes ICP34.5 protein and plays an important role in the process of infecting nerve cells by the virus, has an important role in latent infection of the virus entering a nervous system, and corresponding deletion mutation is made to ensure that the virus enters the nerve cells to form functional deletion of latent infection, meanwhile, on the basis of modifying the mutant Us11 gene in a WR-3 strain, the RL1 gene deletion mutation generates low toxicity, including stably reducing the toxicity to epithelial cells and nerve cells, and ensuring the stability and the irreversible performance of the toxicity attenuation characteristics of the strain under different environments.
Example 2 cloning and purification of WR-1 Virus, WR-2 Virus, WR-3 Virus and WR-4 Virus
In example 1, the WR-1 virus, WR-2 virus, WR-3 virus and WR-4 virus clone purification screening method is as follows:
vero cells were plated in 6-well plates until the cells were fully adherent. The original medium was discarded and washed with PBS. The mutant virus was diluted 10-fold in serum-free MEM medium (1:1-1: 10)6) Different dilutions of virus fluid were then added to each cell well at a volume of 1ml per well and 1 negative control well was set. Placing in 5% CO2Adsorbing at 37 deg.C for 2-3 hr. Subsequently, after discarding the virus solution in the cell culture plate, an agarose medium mixture which had been autoclaved and allowed to cool to about 42 ℃ was slowly added to the cell wells along the plate walls in a volume of 2.5-3 ml/well. Cooling, and adding 5% CO2The culture was inverted at 37 ℃ and observed for 3 to 4 days until plaque appeared.
The plaques appeared visible to the naked eye, and after labeling, the plaques were picked and resuspended in 100ul serum-free DMEM medium and frozen at-80 ℃ for use or continued amplification.
EXAMPLE 3 PCR detection of WR-1 Virus, WR-2 Virus, WR-3 Virus and WR-4 Virus in example 1
After all screened virus clones are proliferated in Vero cells for one generation, 200ul of virus harvest liquid is taken, virus DNA genes are extracted by a general method, the genes are taken as templates to carry out specificity PCR identification of 4 modifying genes, and a PCR system is as follows: forward and reverse primers were 2pmol each, Prime Star Max 25ul, DNA template 2ul, sterile distilled water supplemented to 50ul volume, PCR program: heating at 98 deg.C for 3 min; 98 ℃, 20sec,58 ℃,15sec,72 ℃ for 1 min; 35 cycles. The product was identified by electrophoresis on a 1.2% gel.
The sequence table of the primers used for PCR detection is as follows:
Us5-sense：CTCAGAACCCACCCGAAA
Us5-AS：AAGACAGACT TTGTTATACC
UL41-sense：CCTGTGGAACGTCATGTA
UL41-AS：CTCGTCGTCTTCGTATCC
Us11-sense:ACCATCACCCGAGTCTCT
Us11-AS:GTAGAGCCCTGAGTCATCC
RL1-sense:TAACCTCCACGCCCAACT
RL1-AS:ACGTTAGACCGAGTTCGC
the electrophoresis patterns of the PCR identification results of the WR-1 virus, the WR-2 virus, the WR-3 virus and the WR-4 virus of the examples 1 are shown in the attached figures 1 to 4 respectively through PCR detection.
Example 4 biological characterization of WR-4 Strain (HSV attenuated Strain)
The proliferation characteristics of WR-4 in tissue cultured human diploid epithelial cells WR-N strain and SH-SY5Y cells were initially observed.
The WR-N strain of the human diploid epithelial cell is used as an epithelial cell derived from a human embryonic lung and is used for reflecting the basic biological characteristics that HSV is easy to infect various epithelial cells; SH-SY5Y cell is a neuroblastoma cell from the nervous system, which, although it is a tumor cell, still has the biological characteristics of a nerve cell, which reflects the basic biological properties of HSV infected nerve cells.
WR-4 and the wild strain were first inoculated to the two cells with the same moi, respectively, and the proliferation kinetics of both cells were compared. The experimental results showed that the proliferation kinetics trend of WR-4 on both cells was significantly lower than that of the wild strain (FIGS. 5 and 6). Meanwhile, the morphology of the cloned plaques formed by WR-4 on vero cells is obviously smaller than that of the wild strain (attached figure 7). This result suggests two conclusions. First, the proliferation rate of the virus in epithelial cells is reduced, which suggests that the proliferation rate of the virus is obviously reduced after that of wild strains under the pressure of cell stress response. This indicates that the strategy of modifying Us5 and UL41 has a significant effect. Secondly, the mutant virus strain WR-4 simultaneously shows a lower proliferation rate and a smaller plaque shape in the neuroblastoma SH-SY5Y cells, and the RL1 gene modification related to the infected nerve cells of the strain is suggested to cause the change of the corresponding characteristics of the strain on the infection of the nerve cells.
EXAMPLE 5 preparation of Experimental attenuated live vaccine of WR-4 Strain (HSV attenuated Strain)
The WR-4 experimental attenuated live vaccine strain is detected after proliferation on a single-layer human diploid cell to determine that the virus titer is not lower than 105CCID50Diluted to a viral titer of 10/ml5CCID50The semi-finished product of each ml is subpackaged into 0.5 ml/bottle, and freeze-drying protection is addedAfter the preparation, the vaccine is freeze-dried by a conventional method to obtain the experimental attenuated live vaccine. The mixture was stored at 4 ℃.
EXAMPLE 6 safety observations of mice infected with attenuated live vaccine of WR-4 Strain (HSV attenuated Strain)
The live attenuated vaccine obtained in example 6 was administered at 5X 104CCID50The mice were inoculated by nasal drip, and the progress of infection and pathological manifestations were examined for etiology and pathology within 21 days, while a negative control group (Mock) was set. After inoculation of 20+60 Balb/c mice inoculated with WR-4 strain and 20+60 wild strain (Mckrae strain), 20 infected mice in each of the two groups and 20 mice in the negative control group were weighed every other day and their changes were recorded, and the number of deaths was observed and the survival rate was recorded for 21 days. In addition, 60 of the two groups of infected mice were sacrificed 6 every 2 days and their respective organ tissues were sampled for viral load testing. At the same time, the tissue sections of the major organs including the nervous system were taken for histopathological examination. The results showed that the weight average of 20 mice was steadily increased within 21 days after nasal administration of WR-4 strain to the infected mice (FIG. 8). At the same time, no death occurred (FIG. 9). Whereas 20 mice vaccinated with the wild strain (Mckrae strain) showed significant weight loss and partial individual death after 3-4 days post-infection, the surviving 9 mice began to recover to pre-challenge levels by day 12 (FIGS. 8 and 9). The examination of virus clones in tissue organs of mice sacrificed every 2 days showed that the WR-4 strain and the wild strain showed statistically significant differences in viral load in brain, spinal cord and trigeminal nerve, especially in brain, spinal cord and trigeminal ganglion tissues of mice (FIGS. 10-11), where the wild strain proliferated about 100 times more than 2 orders of magnitude higher than the WR-4 strain. This test suggests that WR-4 has acquired a biological phenotype with greatly reduced proliferative capacity in the body due to genetic defects in the functional protein molecules it encodes that interact specifically with the body's immune system.
Pathological examination of the tissue and organ of WR-4 nose drop infected mice also shows that the pathological damage capability of WR-4 to the main tissue of the mice is greatly reduced (Table 1).
Table 1: histopathological observation of organs of mice infected by mutant strain WR-4 and wild strain
In the central nervous system tissue of the attenuated live vaccine infected mouse, compared with the pathological phenomena of large-scale inflammatory cell accumulation, blood vessel and peripheral tissue congestion, edema, partial nerve cell degeneration and the like caused by wild strain infection. WR-4 strain infected mice showed only mild inflammatory cell infiltration around small vessels. Meanwhile, in the rest tissues, wild strain infection can cause inflammatory cell accumulation, congestion, edema and other manifestations in different degrees, while WR-4 infection only causes slight inflammatory cell infiltration. It is contemplated that obvious cell proliferation manifestations can be seen in the lymph nodes of WR-4 infected animals. These examination results show that the characteristics of WR-4 strain infected mice have been converted to non-pathogenic type, compared to the strongly pathogenic type of wild strain, which shows a marked reduction in the proliferation capacity in mice, without causing significant histopathological damage.
Response characteristics of WR-4 to the in vivo immune system during strain infection in mice:
firstly, the content measurement of interferon including TNF-alpha, IFN-beta and IFN-gamma in the serum of 3 rd, 5 th and 7 th days after mice are infected by wild strains and WR-4 infection shows that the interferon and the IFN-alpha, the IFN-beta and the IFN-gamma in the serum of WR-4 infected animals are obviously different (figure 13), and the IFN-beta and the IFN-gamma in the serum of WR-4 infected animals are obviously higher than those in wild strains infected animals (figure 14 and figure 15). Secondly, the TNF-alpha in the two serums also shows obvious difference, and the wild strain infected animals are obviously higher than the WR-4 infected group (figure 13). In combination with the previously observed apparent proliferation of lymphocytes in lymph nodes upon infection of animals with WR-4, it is believed that the WR-4 strain is capable of directly stimulating the innate immune system with its antigen-associated molecules and producing a corresponding immune response after entry into the mouse, which is in fact a key prerequisite for the activation of the acquired immune response.
EXAMPLE 7 Observation of the immunological protective Effect of WR-4 Strain (HSV attenuated Strain) attenuated live vaccine
The WR-4 treatment group and the wild strain control group were formed by the experimental design of (20+50+ 30). times.2, while 20 negative control groups were set, both of which were first treated with the wild strain (Mckrae strain; 2X 10)4CCID50Per mouse) nose drops, 20 of which were used as the observation groups for weight observation and survival rate, 50 were used as the observation groups for pathology and immune response during infection, and 30 were used as the observation groups for later stage. The WR-4 treatment group starts to administer the inoculation treatment of the WR-4 strain on the 3 rd day after the infection of the wild strain, and performs intramuscular injection once a day for three consecutive days at the dose of 105CCID50In the cases, 20 of the cases were observed from the time of infection with wild strain and WR-4 treatment, and the weight change and death were recorded every other day; the 50 subjects were sacrificed in part 5 at 2 days intervals beginning the day after the 1 st therapeutic injection and ending at 22 days. Organ viral load examination, histological pathology observation, IFN-gamma content examination in blood, and T cell subgroup proportion observation were performed for each sacrificed animal. 30 mice were sacrificed each for 10 mice at 2 months, 4 months and 6 months after infection with the wild strain and administration of WR-4 treatment, and the above indices were observed. All observations indicate that the use of WR-4 as a therapeutic vaccine significantly reduced the clinical manifestations of wild strain infection, significantly shortened the time to recovery of body weight in mice (figure 16), and significantly reduced the mortality (figure 17). During infection, the virus load in each tissue due to wild strain infection was statistically decreased by WR-4 therapeutic immunization (FIGS. 18, 19, 20), while the histopathological damage due to wild strain infection was significantly reduced during WR-4 therapeutic injection (Table 2), and these therapeutic effects were likely to be similar to the increase in interferon levels due to WR-4 therapeutic injection (FIG. 21) and CD8 in T cell population (CD 8)+The subgroup ratios were related to increase (figure 22). More importantly, in later observations, the WR-4 treated group also clearly showed a faster recovery of clinical symptoms caused by the wild strain, especially a reduction in the survival load of the virus in the nervous system and a reduction in survival time (fig. 18, 19, 20). These results suggest that the WR-4 strain constructed by the invention has unique construction strategy, and can inhibit four effectsThe physiological stress of host cells, namely the virus functional genes of a natural immune response system, is subjected to comprehensive deletion mutation, so that the virus strain activates the cell stress system, namely the natural immune response system after entering the cells, the non-specific factors of cell virus resistance, such as interferon expression, are greatly increased, and the strengthened natural immune response is further up-regulated to promote the formation of specific anti-virus immune response without obstacle by removing the inhibition strategy of the virus to an immune presentation link. Therefore, the effect has corresponding therapeutic significance for clinical pathological process expression caused by HSV1 infection of organisms, obviously changes clinical and pathological expression caused by HSV1 infection, and has application significance.
TABLE 2 pathological observations of the mutant WR-4 for various organ tissues after treatment of HSV1 infection
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
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