Hepatitis B treatment vaccine base on inactivated whole recombinant Hansenula polymorpha cells which expresses HBsAg and HBcAg
11191829 · 2021-12-07
Assignee
Inventors
Cpc classification
C12N2730/10134
CHEMISTRY; METALLURGY
A61K39/39
HUMAN NECESSITIES
A61K39/292
HUMAN NECESSITIES
International classification
Abstract
Provided is a hepatitis B treatment vaccine based on an inactivated whole recombinant Hansenula polymorpha cell which expresses HBsAg and HBcAg. An HBsAgVLP and an HBcAgVLP expressed in the recombinant Hansenula polymorpha cell are used as antigens, the amino acid sequence of the HBsAg expressed by the recombinant Hansenula polymorpha contains a total of 19 CTL epitopes, the amino acid sequence of the HBcAg expressed by the recombinant Hansenula polymorpha contains a total of 19 CTL epitopes, and the inactivated whole recombinant Hansenula polymorpha cell is used as an adjuvant.
Claims
1. A hepatitis B therapeutic vaccine comprising an inactivated whole recombinant Hansenula polymorpha cells, comprising and expressing an HBsAg comprising an amino acid sequence of SEQ ID NO: 2 encoded by a nucleic acid sequence of SEQ ID NO: 1 and comprising and expressing an HBcAg comprising an amino acid sequence of SEQ ID NO: 4 encoded by a nucleic acid sequence of SEQ ID NO: 3, wherein the recombinant Hansenula polymorpha cells comprises 15-21 HBcAg-specific CTL epitopes, the HBsAg is adw subtype, the intracellular expression level of the HBsAg in the recombinant Hansenula polymorpha cells is 6-10 μg HBsAg per 10.sup.8 cells, the HBsAg has 16-21 HBsAg-specific CTL epitopes, and wherein the inactivated whole recombinant Hansenula polymorpha cells are an adjuvant, wherein a construction method of an engineering strain of the HBsAg expressed by the recombinant Hansenula polymorpha cells includes the following steps: (1) transforming a plasmid having the DNA sequence as shown in SEQ ID NO: 1 into an URA3-auxotrophic Hansenula cell strain HU-11 (CGMCC No. 1218) of a host cell by cell electroporation, and picking up single colony transformants grown on a MD selection culture plate; (2) selecting colonies with fast growth rate, using semi-quantitative PCR to detect the brightness of HBsAg gene band, selecting colonies with high copy numbers, and screening for 20 to 400 generations in successive subcultures; (3) screening multiple copies of heterologous integrated transformed clones after the successive subcultures in step (2), inducting with methanol for 72 hours, and determining the expression level of HBsAg after disruption of the transformed cells by radioimmunoassay; (4) high-copy, high-expression clones with free plasmids being screened and removed through step (3), and HBsAg gene copy number being detected by quantitative PCR; and (5) basing on the test results of step (4), 30 liters of fermented genetically stabilized recombinant Hansenula HBsAg strain is selected as a primary strain of the engineering strain.
2. The hepatitis B therapeutic vaccine according to claim 1, wherein the HBsAg expressed by the recombinant Hansenula polymorpha cells comprises 19 CTL epitopes, which are shown in from SEQ ID NO:5 to SEQ ID NO:23.
3. The hepatitis B therapeutic vaccine according to claim 1, wherein the HBcAg expressed by the recombinant Hansenula polymorpha cells comprises 19 CTL epitopes as follow, which are shown in from SEQ ID NO:24 to SEQ ID NO:42.
4. The hepatitis B therapeutic vaccine according to claim 1, wherein the host Hansenula polymorpha cell line of the recombinant Hansenula polymorpha cells is HU-11, and the accession number is CGMCC No. 1218, and the disrupted DNA sequence of the orphanin-5-phosphate decarboxylase gene of the host Hansenula polymorpha is shown in SEQ ID NO: 43.
5. The hepatitis B therapeutic vaccine according to claim 1, wherein the dosage form of the hepatitis B therapeutic vaccine is injection solution or lyophilized powder injection.
6. The hepatitis B therapeutic vaccine according to claim 1, the hepatitis B therapeutic vaccine further comprises HBsAg stock solution, or HBcAg stock solution.
7. The hepatitis B therapeutic vaccine according to claim 1, wherein the dosage form of the hepatitis B therapeutic vaccine is pre-filled injection solution.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The present disclosure will become more fully understood from the detailed description and the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
(7) The present disclosure is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Like numbers in the drawings indicate like components throughout the views. As used in the description herein and throughout the claims that follow, unless the context clearly dictates otherwise, the meaning of “a”, “an”, and “the” includes plural reference, and the meaning of “in” includes “in” and “on”. Titles or subtitles can be used herein for the convenience of a reader, which shall have no influence on the scope of the present disclosure.
(8) The terms used herein generally have their ordinary meanings in the art. In the case of conflict, the present document, including any definitions given herein, will prevail. The same thing can be expressed in more than one way. Alternative language and synonyms can be used for any term(s) discussed herein, and no special significance is to be placed upon whether a term is elaborated or discussed herein. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms is illustrative only, and in no way limits the scope and meaning of the present disclosure or of any exemplified term. Likewise, the present disclosure is not limited to various embodiments given herein. Numbering terms such as “first”, “second” or “third” can be used to describe various components, signals or the like, which are for distinguishing one component/signal from another one only, and are not intended to, nor should be construed to impose any substantive limitations on the components, signals or the like.
(9) Development of the Construction of the H. polymorpha Intracellular Plasmid pMPT-02:
(10) The H. polymorpha expression system includes two main components:
(11) (1) A vector system (plasmid) that initiates efficient expression of a foreign gene; and (2) a host cell having a specific selection marker.
(12) 1.5 kb H. polymorpha MOX (methanol oxidase) promoter, 350 bp H. polymorpha MOX (methanol oxidase) terminator, 1.0 kb H. polymorpha autonomous replication sequence HARS, and 1.1 kb Saccharomyces cerevisiae uracil gene ScURA3 were tightly ligated by gene synthesis technology element, and then inserted into the pBluescripII plasmid to construct a shuttle plasmid pMPT-02, which is the applicant's non-exclusive proprietary technology.
(13) Development of the Host Cell Using the Uracil Auxotrophic URA3-Host Cell Line HU-11:
(14) A recombinant H. polymorpha strain HU-11 (The accession number for the deposit: CGMCC No. 1218. The date of the deposit: Sep. 13, 2004. The name and address of the depository: China General Microbiological Culture Collection Center (CGMCC), No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China) in which the orotidine-5-phosphate decarboxylase gene (HURA3) was disrupted by homologous sequence-mediated homologous integration. Compared with the conventional auxotrophic host strains produced by mutagenesis, the recombinant H. polymorpha strain HU-11 has the characteristics of high genetic stability and low back mutation rate. It was convenient for genetic transformation and screening of recombinant strains, and maintains the wild-type strain (ATCC34438). The physiological and biochemical characteristics were beneficial to the culture of recombinant strains and the high expression of foreign proteins, and have high industrial application value. The DNA sequencing result of the disrupted URA3 gene of the H. polymorpha host strain HU11 showed that the five bases of GAAGT were inserted into the 31st base. The insertion of five bases of GAAGT produces a frameshift mutation. The frameshift mutation resulted in a mutation in all of the 254 amino acid codes after the 11th position, and the mutation produced a total of 15 termination codes, indicating that the structural gene of URA3 is no longer re-expressible. The probability that the five bases GAAGT simultaneously produce a back reversion mutation was extremely small. The experimental test also proved that the back mutation rate of the host strain HU11 is zero. This low back-reversion mutation rate of the host strain was particularly advantageous for transformation screening. URA3-ogal deficiency host cell line HU-11 (CGMCC No. 1218) established by gene knock out technology was disclosed in the applicant's previously invention CN1651570A. The DNA sequence in which the disrupted decarboxylase gene (HURA3) is shown in SEQ ID NO: 43.
(15) Preferred Code Gene Design of Recombinant H. polymorpha HBsAg-adw2:
(16) The DNA sequence of HBsAg expression of the recombinant H. polymorpha of the present disclosure is based on the HBsAg adw2 subtypes as shown in SEQ ID NO: 1. The amino acid sequence of the HBsAg is shown in SEQ ID NO: 2.
(17) Construction of the H. polymorpha Intracellular Plasmids pMPT-HBS-Adw and pMPT-HBC:
(18) A synthetic nucleotide sequence according to the sequence of SEQ ID NO: 1 (hereinafter referred to as HBsAg adw2 gene) was constructed into a glycerol strain containing the HBsAg adw2 gene plasmid; the plasmid after correct sequencing was digested with EcoRI/BamHI, and then 701 bp DNA fragment was obtained.
(19) The correct plasmid pMPT-02 was digested with EcoRI/BamHI, and the vector DNA obtained after the gelatinization was ligated to obtain the H. polymorpha intracellular plasmid pMPT-HBS-adw, and the plasmid pMPT-HBS-adw was transformed into E. coli Competent Cell JM109 (Code No. D9052), and then was cultured overnight by plating on. Single colonies were selected from the transformation plates, plasmid DNA was extracted and digested with EcoRI/BamHI, and the results of restriction enzyme digestion showed positive clones. Sequencing confirmed that the plasmid pMPT-HBS-adw was correct.
(20) The HBsAg adw2 gene was inserted into the multiple cloning site of the H. polymorpha expression system intracellular plasmid pMPT-02: between EcoRI and BamHI. The full length of the plasmid pMPT-HBS-adw was 7665 bp. A schematic diagram of the construction process of plasmid pMPT-HBS-adw was shown in
(21) The plasmid pMPT-HBC based on the sequence shown in SEQ ID NO: 3 was constructed in the same method as in the construction of plasmid pMPT-HBS-adw. The physical map of the pMPT-HBC adw2 plasmid was shown in
(22) Construction of the Recombinant H. polymorpha HBsAg Engineering Strain and the Recombinant H. polymorpha HBcAg Engineering Strain:
(23) In order to construct the recombinant H. polymorpha hepatitis B virus surface antigen (HBsAg) adw2 subtype engineering strain, the cell electroporation technology developed by the applicant was applied. The RC pulse: amplitude 1500V, capacitance 22 μf, and time constant 3-5 ms electric shock 1 time, adopted the pMPT-HBS-adw plasmid transformed into H. polymorpha cells of the HU-11 strain (CGMCC No. 1218) from which the URA3-gene was knocked out. The single colony transformants were picked up on the MD selection culture plate and transferred to the MD liquid medium for continuous subculture. The adw2 subtype HBsAg gene and the corresponding regulatory components were multi-copy and heterologously integrated into the host H. polymorpha cell chromosome. After a single colony of more than one thousand transformant single colonies, the following three steps were screened:
(24) (1) Clonal strains with large single colonies and fast cell growth have a high probability of multiple copies.
(25) (2) The PCR technique was used to compare the electrophoretic band luminance of the HBsAg gene and the single copy number MOX (methanol oxidase) gene, and the HBsAg gene copy number was determined semi-quantitatively.
(26) (3) The expression level of HBsAg released after methanol-induced and shake flask culture for 72 hours was detected.
(27) The application of PCR technology to transformants screening was a new creation of this application. The multiple copies of the foreign gene HBsAg are determined and heterologously integrated in the H. polymorpha chromosome, while the MOX gene in the H. polymorpha chromosome was intact and not destroyed. They all play an important role and show unique advantages of the H. polymorpha expression system. A pair of primers were designed to simultaneously amplify the MOX gene (single copy) and the heterologous integrated HBsAg foreign gene (multicopy) in the H. polymorpha chromosome. By comparing the brightness of the bands of the amplified product in agarose gel electrophoresis, it was possible to roughly determine whether the HBsAg gene was multiple copies. This method was used for the preliminary screening of multi-copy strains of engineered HBsAg gene. The amplified HBsAg fragment was 800 bp in length and the amplified MOX fragment was 2000 bp in length.
(28) PCR product agarose gel electrophoresis: the amplified product of HBsAg gene of engineering strain was about 800 bp, and the amplification product of H. polymorpha single copy gene MOX gene was about 2000 bp. The recombinant H. polymorpha hepatitis B virus surface antigen (HBsAg) engineering strain obtained by final screening is numbered as HS604-5.
(29) Using the plasmid pMPT-HBC, the electrophoresis photograph of the PCR amplification product of the engineered strain obtained via the same method by screening from over 100 copies transformant was shown in
(30) Determination of Intracellular HBsAg VLP Expression in Recombinant H. polymorpha HBsAg Adw2 Subtype Engineering Strain Fermentation Broth:
(31) 10 μg of adw2 subtype HBsAg hepatitis B surface antigen lyophilized standard provided by Tiantan Biotechnology was diluted with diluent to dilute to 1024 ng/mL, 512 ng/mL, 256 ng/mL, 128 ng/mL, 64 ng/mL, 32 ng/mL, 16 ng./mL, 8 ng/mL, 4 ng/mL, 2 ng/mL, Ong/mL (diluent) a total of 11 standard points, and using radioimmunoassay kit to detect HBsAg reaction.
(32) The obtained engineering strain (No. HS604-5) was subjected to 30 liters of pilot fermentation (batch number 20150422), and 1 mL 10 OD.sub.600 nm was sampled after 87 hours of fermentation. After disrupting the cells with glass beads (cell disruption rate: 65%), the 200-fold diluted sample and the standard were separately reacted in the radioimmunoassay kit at the same time, and the expression of HBsAg antigen obtained by the γ-counter auto-completed curve was 126.9 (ng/mL). Based on the above, the expression level of intracellular HBsAg antigen in recombinant H. polymorpha was calculated as:
126.96 (ng/mL)×200÷10×4.0×10.sup.7×65%/mL=9.8 μg/10.sup.8 cells
(33) An electron micrograph of the recombinant HBsAg pure stock solution of recombinant H. polymorpha was shown in
(34) The expression of HBcAgVLP in the fermentation broth of recombinant H. polymorpha HBcAg engineering strain determined by the same method was 8-12 μg/10.sup.8 cells.
(35) Expression of Virus-Like Particles (VLP) in Recombinant H. polymorpha HBcAg Cells
(36) The central rule of modern molecular biology is: DNA.fwdarw.RNA.fwdarw.amino acid primary sequence.fwdarw.protein molecule high structure.fwdarw.protein molecular function; symbol.fwdarw.indicates: decision.
(37) Different genotypes of hepatitis B virus core antigen HBcAg protein molecule is composed of 183 or 185 amino acid residues to form a primary sequence, which determines the secondary structure, tertiary structure and quaternary structure of HBcAg.
(38) Experiments show that HBcAg isolated from isolated HBV particles, infected cells, recombinant E. coli or recombinant yeast were found to be assembled into two sizes of particles: The T=3 type consists of 180 identical protein subunits with a molecular weight of 21 kD (i.e. 90 homo-dimers), with a diameter of 30 nm, and 90 spikes on the 4 surfaces. The T=4 type consists of 240 identical protein subunits with a molecular weight of 21 kD (i.e. 120 homo-dimers), with a diameter of 34 nm, and 120 spikes on the surface.
(39) The transmission electron micrograph of the crude extract of the cell suspension of HBC-40-25 engineering strain of the present disclosure clearly shows HBcAg virus-like particles.
(40) The above-described recombinant H. polymorpha HBcAg engineering strain (HC-40-25, 172 amino acids) with lacking amino acid is compared with six strong positive strains HBC-17 of recombinant H. polymorpha HBcAg (183 amino acids) with full-length amino acid. (1 OD.sub.600 nm/ml cells induced by methanol culture for 3 days, disrupted by glass beads, 1000-fold diluted sample), and the immunogenicity (unit: NCU/mL) was tested as follows:
(41) TABLE-US-00001 HBC-40-25 HBC-17 HBC-31 HBC-36 HBC-39 HBC-41 HBC-51 35.21 5.83 5.64 5.64 6.34 8.99 5.75
(42) The immunoreactivity of HBC-40-25 strain was more than three times that of the other six strong positive strains.
(43) Optimization of Heat-Inactivated Recombinant H. polymorpha HBsAg and HBcAg Cell Conditions:
(44) In order to determine the optimal conditions for the inactivated recombinant H. polymorpha, the following requirements should be met: (1) Reduce the survival rate of the inactivated recombinant H. polymorpha less than 5%. (2) Maintain a complete cellular structure to avoid leakage of heat-inactivated recombinant H. polymorpha intracellular antigenic substance. (3) Maintain the thermal stabilities of HBsAg virus-like particles (VLP) and HBcAg virus-like particles (VLP) expressed in recombinant H. polymorpha cells, so as to avoid the antigenicity decline. The above three requirements are the main condition of the production for the heat-inactivated recombinant H. polymorpha expressing HBsAg and HBcAg, and would provide the basis for the development of vaccine manufacturing and verification procedures. In addition, the thermal stability of HBsAg virus-like particle (VLP) expressed in recombinant H. polymorpha cells is the first issue to be solved. For this purpose, 16 sets of different temperature (50° C., 52° C., 54° C., 56° C. and 58° C.), and different time (1 hr, 2 hr, and 3 hr) of the heat-inactivation test of recombinant HBsAg H. polymorpha cells were designed and set at 20° C. as the control group. During the detection: as the heat inactivation temperature and time increase, the solubility of the outer layer of the cell wall increases, the cell breakage rate increases, and the intracellular HBsAg VLP antigen reactivity peak multiplied at 52° C., 1 hr, while the intracellular HBcAg VLP antigen reactivity peak multiplied at 56° C., 1 hr. The extracellular HBsAg and HBcAg antigens reactivity was extremely low during the temperature and time changes of the heat-inactivation assay, indicating that intracellular HBsAg VLP did not leak out, maintaining the heat-inactivated recombinant H. polymorpha cell structure. The survival rate of heat-inactivated cells was as low as 1/50,000 at 56° C., 3 hr. Therefore, a basis for optimizing the conditions for heat inactivation of recombinant H. polymorpha cells was provided.
(45) The HBsAg-Specific and HBsAg-Specific CTL Cells Trigger Reversal of Immunity without Cell Damage
(46) The results of a series of studies on hepatitis B virus infection in chimpanzees indicated that, the HBV-specific CD8+ T cells, which produce INF-γ and target to hepatocytes infected with HBV; in the first step is to reduce the pool of cccDNA molecules by more than 90% without cell damage, and the second step is to improve the process of destroying infected liver cells and trigger reversal of immunity.
(47) Based on three experiments described CTL epitopes review, prediction and patented invention reported, HBV capsid antigen (Pre-S1-Pre-S2-HBsAg) has 23 CTL epitopes that did not repeat. The amino acid sequence of the HBsAg antigen of the present disclosure includes the following 19 CTL epitopes: VLQAGFFLL (SEQ ID NO: 5), PFVQWFVGL (SEQ ID NO: 6), FLLTRILTI (SEQ ID NO: 7), WYWGPSLYSI (SEQ ID NO: 8), SLNFLGGSPV (SEQ ID NO: 9), FLGGSPVCL (SEQ ID NO: 10), LYSIVSPF (SEQ ID NO: 11), LYSIVSPFI (SEQ ID NO: 12), PFIPLLPIF (SEQ ID NO: 13), LLLCLIFLL (SEQ ID NO: 14), LLCLIFLLV (SEQ ID NO: 15), LLDYQGMLPV (SEQ ID NO: 16), LVLLDYQGML (SEQ ID NO: 17), VLLDYQGML (SEQ ID NO: 18), WLSLLVPFV (SEQ ID NO: 19), LLVPFVQWFV (SEQ ID NO: 20), GLSPTVWLSA (SEQ ID NO: 21), SIVSPFIPLL (SEQ ID NO: 22), and LLPIFFCLWV (SEQ ID NO: 23).
(48) The amino acid sequence of the HBcAg antigen of the present disclosure includes the following 19 CTL epitopes: SFLPSDFF (SEQ ID NO: 24), FLPSDFFPSI (SEQ ID NO: 25), DFFPSIRDLL (SEQ ID NO: 26), FFPSIRDLL (SEQ ID NO: 27), SYVNVNMGL (SEQ ID NO: 28), SYVNVNMGLKI (SEQ ID NO: 29), YVNVNMG (SEQ ID NO: 30), YVNVNMGLK (SEQ ID NO: 31), WFHISCLTF (SEQ ID NO: 32), CLTFGRETV (SEQ ID NO: 33), VLEYLVSFGV (SEQ ID NO: 34), EYLVSFGVW (SEQ ID NO: 35), EYLVSFGVWI (SEQ ID NO: 36), AYRPPNAPI (SEQ ID NO: 37), AYRPPNAPIL (SEQ ID NO: 38), APILSTLPE (SEQ ID NO: 39), ILSTLPETTV (SEQ ID NO:40), STLPETTVVRR (SEQ ID NO: 41), and RGRSPRRRTP (SEQ ID NO: 42).
(49) H. polymorpha Recombinant Hepatitis B Vaccine Product is Preferred as Prefilled Injection:
(50) The routinely dispensed recombinant hepatitis B vaccine (yeast) pre-filled syringe was processed thermal stability test at 37° C. for 45 days, and has proved that the relative in vitro relative efficacy (RP) of the vaccine met the requirements, while at the same storage condition, RP of routinely dispensed hepatitis B vaccine did not meet the requirements. Pre-filled syringe-packed recombinant hepatitis B vaccine (yeast) can be transported without refrigerated in a short time, stored and used.
(51) The pre-filled syringe has 1 needle and 1 box, which is easy to use, easy to learn to use and essentials, a disposable syringe cannot be reused. Vaccination without a separate syringe can prevent from infection or infectious diseases spread caused by not completely sterilized glass syringe, the adverse effects caused by improper needle selection or the risk of disposable syringe being reused.
(52) Full vaccination of pre-filled hepatitis B vaccine syringe has a good comprehensive cost-benefit ratio.
(53) The following embodiments are intended to be illustrative, and not restrictive, and the scope of the present disclosure is not limited by the following embodiments.
First Embodiment
(54) The pMPT-HBS-adw plasmid was constructed based on the sequence of SEQ ID NO: 1 (an expression vector comprising the sequence of the SEQ ID NO: 1). The construction of plasmid pMPT-HBS-adw includes the following steps:
(55) The HBsAg adw2 gene was synthesized according to the DNA sequence of SEQ ID NO: 1; and the glycerol strain containing the HBsAg adw2 gene plasmid was constructed and named as MC407B-16.
(56) The correctly sequenced MC407B-16 plasmid was digested with EcoRI/BamHI, and the digested product was used a TaKaRa PCR Fragment Recovery Kit (Code No. D301) to recover 701 bp DNA fragment called Inset DNA6.
(57) The correct plasmid pMPT-02 was digested with EcoRI/BamHI, and the vector DNA obtained from the DNA recovery kit was called Vector DNA6.
(58) Inset DNA6 was ligated to Vector DNA6 by using Solution of the TaKaRa DNA Ligation Kit (Code No. D6022), and then thermally transformed into E. coli Competent Cell JM109 (Code No. D9052), and the cells were plated in the transformation plate and cultured overnight. Single colonies were selected from the transformation plate, and plasmid DNA was extracted. The plasmid DNA was digested with EcoRI/BamHI. The results showed that MC407A+B+C+D-77˜80 were positive clones.
(59) The plasmid MC407A+B+C+D-77 was sequenced respectively with primer RV-M, M13-47, MC407P1, MC407P2, MC407P3, MC407P4, MC407P5, MC407P6, MC407P7, MC407P8, MC407P9, MC407BF11, MC407BR11 to prove the plasmid pMPT-HBS-adw were correct.
Second Embodiment
(60) Construction of a Recombinant H. polymorpha HBsAg Engineered Strain.
(61) Recombinant Hepatitis B Vaccine H. polymorpha transformation and screening illustration: The transformation and screening process of the recombinant H. polymorpha was shown in
(62) Specifically
(63) 1) The pMPT-HBS-adw plasmid was transformed into the URA3-auxotrophic H. polymorpha cell strain HU-11 (CGMCC No. 1218) of the host cell by cell electroporation. The culture medium was selected using a selection medium (MD liquid medium). The single colony transformants were picked up on the MD selection culture plate and transferred to the MD liquid medium for continuous subculture. The adw2 subtype HBsAg gene and the corresponding regulatory components were multi-copy and heterologously integrated into the host H. polymorpha cell chromosome.
(64) 2) Strain screening included the following steps (1) Selecting a single colony of uracil prototrophic transformants Colonies with rapid growth rate of bacteria were selected. PCR was used to detect the brightness of HBsAg gene bands. Colonies with a large number of copies were selected, and single colonies were shake-cultured in a selective medium, and successively subcultured for 20 to 400 generations; (2) Screening multiple copies of heterologous integrated transformed clones After subculture in step (1), and 72 hours of methanol-induced culture, the expression level of HBsAg released by the disruption of transformant cells was determined by radio immunoassay or radioimmunoassay (RIA); (3) Screening out high-copy, high-expression clones of free plasmids The clones screened by step (2) were cultured in YPD complete medium for 48 hours, and then transferred into a selection medium plate for cloning culture, and the HBsAg gene copy number was detected by quantitative PCR, and the expression level of HBsAg was detected by RIA. (4) Based on the detection result of the step (3), the primary strain of the genetically stabilized recombinant H. polymorpha HBsAg engineering strain was selected.
(65) The recombinant H. polymorpha HBcAg engineering strain was constructed and screened in the same method. Third Embodiment 30 liters of pilot fermentation (The recombinant H. polymorpha HBcAg engineering and the recombinant strains of H. polymorpha
(66) HBsAg engineering strain fermentation process using the same method)
(67) The main process:
(68) 1) Strain stored in liquid nitrogen was thawed by 200 ml seed medium, inoculated into the medium, divided into two 0.5 L shake flasks, and cultured at 31° C. for 22 hours as a first-class seed;
(69) 2) The primary seed was transferred into the secondary seed culture medium with 1600 ml seed medium, divided into six 1 L shake flasks, and incubate at 31° C. for 20 hours as a secondary seed;
(70) 3) 12 L fermentation medium was adjusted to pH 5.5 and transferred into a 30 L fermenter, and then the secondary seed was inoculated under growing at 30-31° C. through two sources of glycerol and methanol; growth, de-repression and induction for the three phases, and co-culture 85 to 96 hours, the cells were harvested after 2-3 hours stopped induction. The frozen cells are homogenized.
(71) Operation Points:
(72) (1) The feeding operation of the growth phase was going when the dissolved oxygen was consumed and the basal medium was consumed; the flow acceleration was gradually increased as the consumption of the basic medium increases, and the flow was added before 2-3 hours the dissolved oxygen was recovered.
(73) (2) In the later stage of the growth phase, pay attention to the dissolved oxygen recovery, record the lowest value of dissolved oxygen, and start to flow when the dissolved oxygen rises to 70-80%, and enter the de-repression phase.
(74) (3) After the later stage of the de-repression phase, the dissolved oxygen began to rise after the end of the flow. When the dissolved oxygen was raised to 70-80%, the methanol induction solution was added, and the methanol concentration is controlled at 3-5%0; the flow acceleration was controlled by the methanol detection flow controller.
(75) (4) Stopping methanol addition before 2-3 hours the end of fermentation to reduce methanol residue during cell harvest.
(76) Medium
(77) 1. Preparation of Calcium Chloride Solution 11.33 g CaCl.sub.2 was accurately weighed and put it into a cleaned triangular flask, deionized water was appropriately added to dissolve and dilute to 200 ml.
(78) 2. Preparation of Micro Element Solution
(79) Accurately weighting the following reagents:
(80) TABLE-US-00002 (NH.sub.4).sub.2Fe(SO.sub.4).sub.2•6H.sub.2O 1000 mg CuSO.sub.4•5H.sub.2O 80 mg ZnSO.sub.4•7H.sub.2O 300 mg MnSO.sub.4•H.sub.2O 400 mg EDTA 1000 mg
(81) The weighed reagent was placed in a cleaned triangular flask, dissolved in deionized water and dissolved to 200 ml.
(82) 3. Preparation of Vitamin Solution
(83) Accurately weighting the following reagents:
(84) TABLE-US-00003 d-Biotin 6 mg Thiamin HCl 2000 mg
(85) Biotin was first dissolved in 10 ml of 50% isopropanol, and then dissolved in Thiamin HCl, and then dissolved in an appropriate amount of deionized water to a volume of 100 ml.
(86) 4. Preparation of Trace Element Solution
(87) Accurately weighting the following reagents:
(88) TABLE-US-00004 NiSO.sub.4•6H.sub.2O 10 mg CoCl.sub.2•6H.sub.2O 10 mg H.sub.3BO.sub.3 10 mg Na.sub.2MoO.sub.4•2H.sub.2O 10 mg KI 10 mg
(89) The weighed reagent was placed in a cleaned triangular flask, and an appropriate amount of deionized solution was added to a volume of 50 ml.
(90) The above four solutions were separately sterilized and filtered for use.
(91) 5. Preparation of Seed Salt Solution
(92) Accurately weighting the following reagents:
(93) TABLE-US-00005 NH.sub.4H.sub.2PO.sub.4 80 g MgSO.sub.4•7H.sub.2O 18 g KCl 20 g NaCl 2 g
(94) The weighed reagent was placed in a cleaned triangular flask, dissolved in deionized water and dissolved to a volume of 1600 ml.
(95) 6. 27 g of glycerin was weighted in a 2000 ml flask, mixed with a salt solution of 360 mL, and made up to 1800 ml with deionized water. The same amount was dispensed into two 2000 ml flasks, and autoclaved at 110° C. for 30 minutes.
(96) Two empty 500 ml triangle bottles, six 1000 ml triangle bottles, a 100 ml graduated cylinder and a 500 ml graduated cylinder all were sterilized under 110° C., 30 minutes high pressure steam.
(97) 7. Primary Seed Medium
(98) In the clean bench, 100 ml of each sterilized glycerin solution was taken aseptically, and added separately into two 500 ml sterilized flasks, and respectively added the following:
(99) TABLE-US-00006 Calcium chloride solution 1 ml Micro element solution 1 ml Vitamin solution 0.5 ml Trace element solution 0.25 ml
(100) Shaking the above solution.
(101) 8. Secondary Seed Medium
(102) 1600 ml of sterilized glycerin solution was placed in a clean bench with sterile operation technique and placed in a 2000 ml sterilized triangle, and separately added:
(103) TABLE-US-00007 Calcium chloride solution 16 ml Micro element solution 16 ml Vitamin solution 8 ml Trace element solution 4 ml
(104) 9. Fermentation Medium
(105) The following reagents were accurately weighted and dissolved in 2000 ml of deionized water.
(106) TABLE-US-00008 NH.sub.4H.sub.2PO.sub.4 175 g MgSO.sub.4•7H.sub.2O 40 g KCl 44 g NaCl 4.4 g
(107) 520 g glycerin was weighted and added into a small 500 ml beaker. 10 ml defoamer was added into the beaker to sterilize, and then added:
(108) TABLE-US-00009 Calcium chloride solution 175 ml Micro element solution 175 ml Vitamin solution 88 ml Trace element solution 44 ml
(109) 10. Feed Medium
(110) 87 g NH.sub.4H.sub.2PO.sub.4, 260 g glycerin and 500 ml deionized water were added into 1000 ml flask, and then wrapped feed line and sterilized at 110° C. for 30 minutes.
(111) 11. De-Repression Solution
(112) 1800 g glycerin and 660 ml deionized water were added into a 5000 ml flask, and then wrapped feed line and sterilized at 110° C. for 30 minutes. 540 ml filter-sterilized salt solution was added after cooling.
(113) 12. Induction Solution
(114) 400 ml glycerin was added into a 1000 ml flask, and then wrapped feed line and sterilized at 110° C. for 30 minutes. 1600 ml methanol was added aseptically after cooling.
Fourth Embodiment
(115) Purification
(116) The recombinant H. polymorpha HBsAg engineering strain fermentation broth obtained from the third embodiment was harvested and the cells were washed. The detailed steps of purification can be found in References: Li Jin, Kong Yan. Recombinant Hepatitis B Vaccine Production Process. See Li Jin, Yu Yu, Dong Dexiang Editor: Biopharmaceutical Equipment And separation and purification techniques. 1st edition. Beijing: Chemical Industry Press, 2003: 348-349. The harvested cells can be crushed by a homogenizer to release HBsAg; the cell debris was removed by filtration with a 0.22 μm microporous filter; the small molecular impurities were removed by ultrafiltration with a 300K ultramicrofilter; and the HBsAg was extracted by silica gel adsorption treatment. Finally, it was purified by butyl agarose hydrophobic chromatography.
Fifth Embodiment
(117) The Optimal Operating Condition Tests of the Inactivated Recombinant H. polymorpha Cell.
(118) In order to determine the optimal conditions for the inactivated recombinant H. polymorpha, the following requirements should be met:
(119) (1) Reduce the survival rate of the inactivated recombinant H. polymorpha less than 5%.
(120) (2) Maintain a complete cellular structure to exert as an adjuvant with multi titer activity of the inactivated recombinant H. polymorpha.
(121) (3) Maintain the virus-like particle (VLP) expressed in the inactivated recombinant H. polymorpha intact, so as to avoid the antigenicity decline.
(122) The above three requirements are the main condition of the production for the inactivated recombinant H. polymorpha expressing HBsAg and HBcAg, and would provide the basis for the development of vaccine manufacturing and verification procedures. In addition, the thermal stability of the virus-like particle (VLP) expressed in the inactivated recombinant H. polymorpha is the first issue to be solved.
(123) (1) Preparation of the Inactivated Recombinant H. polymorpha Cell with Optimal Operating Condition.
(124) After the H. polymorpha engineering strain (strain number HS604-5) was cultured by fermentation or shake flask induction, the cells were washed with phosphate buffered saline (PBS) for three times by centrifugal process, and suspended the H. polymorpha in PBS for the volume calculation. The cells were counted using OD.sub.600 nm, diluted to 10 OD.sub.600 nm/ml with PBS, 2 ml per tube; each test group was provided with two test tubes which were disrupted group and not disrupted group, respectively; 16 test groups were required to prepare 32 tube sample tubes.
(125) Place them in a set temperature water bath and thermally inactivate the recombinant H. polymorpha for a set time.
(126) The inactivated recombinant H. polymorpha should be cultured for 3 days at 37° C. in a chloramphenicol complete medium agar dish, and the survival rate was counted. The inactivated H. polymorpha is stored at 4° C. for further use.
(127) (2) H. polymorpha recombinant HBsAg (HS604-5 strain) cell heat-inactivation test group was established:
(128) 20° C. room temperature group 1 tube (control)
(129) TABLE-US-00010 50° C. 1 hour 2 hours 3 hours 52° C. 1 hour 2 hours 3 hours 54° C. 1 hour 2 hours 3 hours 56° C. 1 hour 2 hours 3 hours 58° C. 1 hour 2 hours 3 hours
(130) Total is 16 test groups. After inactivation, the HBsAg antigen activity was detected by radioimmunoassay HBsAg reagent; 1:100 dilutions and 1:1000 dilutions, and double tubes were set. The test results were used to analyze and determine the optimal process conditions for the inactivation of H. polymorpha in this new hepatitis B vaccine.
(131) (2) The heat-inactivation test groups of the recombinant H. polymorpha HBcAg engineering strain were established:
(132) 20° C. room temperature group 1 tube (control)
(133) TABLE-US-00011 50° C. 1 hour 2 hours 3 hours 52° C. 1 hour 2 hours 3 hours 54° C. 1 hour 2 hours 3 hours 56° C. 1 hour 2 hours 3 hours 58° C. 1 hour 2 hours 3 hours
(134) Total is 16 test groups. After heat-inactivation, the HBcAg antigen activity was detected by radioimmunoassay HBcAg reagent; 1:100 dilutions and 1:1000 dilutions, and double tubes were set. The test results were used to analyze and determine the optimal process conditions for the inactivation of H. polymorpha in this new hepatitis B vaccine.
REFERENCES
(135) 1. Qi Xiaoqiu, etc., the national population of hepatitis B virus epidemiology investigation report, the first edition of April 2011, People's Health Publishing House. 2. Bowen D G et. al, Intrahepatic immunity: a tale of two sites? Bowen D G et. al, Trends Immunol. 2005, 26(10):512-7. 3. Thomas H. King1, et. al, A Whole Recombinant Yeast-Based Therapeutic Vaccine Elicits HBV X, S and Core Specific T Cells in Mice and Activates Human T Cells Recognizing Epitopes Linked to Viral Clearance, 2014, POLS. 4. Haibin Huang et. al, Robust Stimulation of Humoral and Cellular Immune Responses following Vaccination with Antigen-Loaded β-Glucan Particles, 2010, MBio.asm.org, 1(3): 1-7. 5. Robert Thimme et. al, CD8+ T Cells Mediate Viral Clearance and Disease Pathogenesis during Acute Hepatitis B Virus Infection, JOURNAL OF VIROLOGY, 2003, p.68-76. 6. Stefan F. Wieland et. al, Expansion and contraction of the hepatitis B virus transcriptional template in infected chimpanzees. Proc Natl Acad Sci USA. 2004 Feb. 17; 101(7): 2129-2134. 7. John M. Murray et. al, Dynamics of hepatitis B virus clearance in chimpanzees, 2005 Dec. 6; Proc Natl Acad Sci USA. 102(49): 17780-17785. 8. Thimme R et. al, CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection; J Virol. 2003 January; 77 (1):68-76. 9. Zeng Zhu-tian, Liver-induced systemic immune tolerance and its reversal, Ph.D thesis, the University of Science and Technology of China, 2014. 10. Applicant: Fudan University, Vaccine for controlling persistent infection of hepatitis B virus, 2009, Publication No. CN102038948A. 11. Florian K Bihl et. al, Simultaneous assessment of cytotoxic T lymphocyte responses against multiple viral infections by combined usage of optimal epitope matrices, anti-CD3 mAb T-cell expansion and “RecycleSpot”; Journal of Translational Medicine 2005, 3: 20 1-19. 12. Yuji Sobao et. al, Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia, Journal of Hepatology, 2001, 34: 922-929. 16. Applicant: Yuzhang Wu et al., Immunogen for preparation of therapeutic vaccines or drugs for treatment of hepatitis B and the producing method and use thereof, Publication No. CN1483736A. 17. Deng Xiaoyan, Research on Human Genome-wide Hepatitis B Virus Gene (Sub) Type Recombinant, Ph.D thesis of Chongqing Medical University, May 2012.
(136) The foregoing description of the exemplary embodiments of the disclosure has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the disclosure to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
(137) The embodiments were chosen and described in order to explain the principles of the disclosure and their practical application so as to enable others skilled in the art to utilize the disclosure and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present disclosure pertains without departing from its spirit and scope.