Studies On Production Of M2e Of Influenza A Virus And Protective Immunity Against Influenza A Virus

Influenza virus is the pathogen of influenza, which often swept the global and is a kind of highly contagious disease with the high lethality rate among all of the infectious diseases. The best way to prevent influenza virus at the present time is vaccination. All commercial human influenza vaccines presently available contain hemagglutinin and neuraminidase as their main viral antigen. Influenza hemagglutinin and neuraminidase, are large glycoproteins, well accessible on the viral membrane and highly immunogenic which can induce anti-HA and anti-NA in host. Although they can offer fine protection, they have one considerable drawback: antigenic drift and antigenic shift which cause the variation of influenza virus and may result in a pandemic. The conventional influenza vaccine must be adapted almost every year to follow the antigenic drift and shift of the virus and the antibodies exist in human body induced by vaccination or exposure to previously circulating virus could not efficiently prevent the infection of mutated strains. It is much inconvenient for influenza vaccine preparation, and if vaccine doesn't match with prevalent strain, it can't produce enough immunoprotection.A universal vaccine, which protects effectively against all influenza A strains and provides long-lasting immunity, will definitely be an attractive solution to overcome the threat of this promiscuous virus. Except HA and NA, the matrix protein M2 is also a transmembrane protein of the influenza A virus. Although the protein plays an important role in virus replication, it is much smaller, only 97 amino acids long.Among these amino acids, 24 amino acids at the N terminus are exposed at the membrane surface, 19 amino acids span the lipid bilayer, whereas the remaining 54 residues are located on the cytoplasmic side of the membrane. The extracellular domain of the influenza M2 protein (M2e) remained nearly invariable since the first human influenza A virus strain A/WS/33(H1N1) was isolated in 1933. Several studies have demonstrated that influenza vaccines based on M2e exhibited effective protective immunity, importantly, this protective immunity caused by M2e is broad-spectrum, make it become one of the important target genes in the study of general vaccine.Human serum albumin (HSA) is the majority component of serum, which is involved in maintenance of osmolarity and plasma volume. It consists of a single non-glycosylated polypeptide chain of 585 amino acids forming a globular protein. It is a natural carrier involved in the endogenous transport and delivery of various natural as well as therapeutic molecules and has a long half-life of 19 days in human beings. After fusing with human serum albumin, the fusion protein can reduce its bioavailability and prolong its half-time in vivo to boost therapeutic effect. Meanwhile, HSA could achive high-level expression in P. pastoris and hybrid protein was less in supernatant so it was easy to achieve large-scale production.However, M2e could only induce much lower immunologic reaction since it is a small peptide of 23 amino acids and the molecular weight is small. In this study, we aim to modify pPICZαC-HSA as a vaccine vector and link the M2e-peptide to HSA. Our strategy is to use HSA to boost the immunogenicity of M2e peptide.The contents of this paper are focused on the following parts:â‘ Constructing expression vector pPICZαC-HSA/M2e, transforming it into Pichia pastoris via electroporation and screening the engineering strains secreting the protein at high levels steadily.â‘¡Studying the large-scale fermentation process of HSA/M2e in 80L fermentor, optimizing the main parameters, and creating a new method for large-scale purification of HSA/M2e.â‘¢Purified HSA/M2e as a vaccine was administered to mice and observed the M2e-specific immune response induced by HSA/M2e and its protection against influenza virus challenge.1. Construction of Pichia pastoris expression system for HSA/M2e:1.1 Construction expression vector pPICZαC-HSA/M2e:HSA gene was obtained by RT-PCR from liver, then it was inserted into pMD18-T vector. Results of DNA sequence analysis of the recombinant vector pMD18-T-HSA demonstrated that cDNA encoding human HSA was correctly inserted into pMD18-T vector and the sequence was consistent with that logged in GenBank (accession No. NM₀00477). After confirmation by sequencing, BstbI and kpnI target sites were added using expression primers by PCR. After digestion with BstBI and KpnI, it was ligated into vector pPICZαC which was also digested with BstBI and KpnI. The plasmid was named pPICZαC-HSA. Two oligonucleotides of M2e were synthesized which had restriction endonuclease cohesive ends of Eco81I and KpnI, and DNA fragment after two oligonucleotides annealing was ligated into vector pPICZαC-HSA which was digested with Eco81I and KpnI. The plasmid was named pPICZαC-HSA/M2e. After confirmation by endonuclease digestion assay and sequencing, the expression vector pPICZαC-HSA/M2e was linearized and transformed into Pichia pastoris X-33 via electroporation.1.2 Screening and identification of the transformed Pichia pastorisTransformed yeasts were plated on YPD plates containing Zeocin to isolate Zeocin -resistant clones. After clones were cultured for 72 hours at 30℃, 10 single clones were picked into culture. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the expression primers. Yeast clones tested positively by PCR were proliferated and then HSA/M2e was induced with 0.5% methanol. The supernatant of fermentation was identified by SDS-PAGE and Western Blot and the highest level strain was screened. Western Blot results indicated that the recombinant HSA/M2e was identical with HSA and M2e. And the HSA/M2e protein was purified by AKTA explorer using Mono S cation-exchange chromatography, ultrafiltration and Mono Q anion-exchange chromatography. The purity coefficient of HSA/M2e could be more than 95% and amino acid sequencing could prove the 9 N terminal amino acid were identical with natural HSA.2. Studies on large-scale fermentation and purification process of HSA/M2e2.1 Studies on large-scale fermentation process of HSA/M2eThere are many advantages as a kind of expression host of Pichia pastoris, and it's very suitable for large-scale expression of the extraneous proteins. We further explored the large-scale fermentation process of HSA/M2e in 80L fermentor. The study was focused mainly on pH value, culture medium, dissolved oxygen, methanol feeding speed, initial biomass and etc. We could confirm that the best conditions for fermentation were as follows: in the FM21 medium (including 0.5% peptone) of pH 5.5, when a cell yield of 200 g/L wet weight was achieved, methanol induction phase began. In the fermentation broth of pH5.5, DO between 20³0% and the supply speed of methanol is 11ml/h/L initial fermentation volume, after methanol induction for about 42 h the expression level of HSA/M2e peaked. The concentration of HSA/M2e in the broth can reach 1080 mg/L.2.2 A new method to purify HSA/M2e at large-scaleThe fermentation supernatant was purified with SP Sepharose XL cation exchange chromatography and sourceTM 30 RPC hydrophobic chromatography, then the eluate was distilled to condense HSA/M2e. The degree of purity could be more than 93% and the yield coefficient was higher than 65%.3. Studies on animal vaccination with HAS/M2e and challenge experiments3.1 study on animal vaccination with HSA/M2e6⁸-week-old male BALB/c mice were divided into 3 groups randomly, 9 mice per group. We gave a total of three injections intraperitoneally, at 2-week interval. PBS, HAS/M2e and inactivated split influenza vaccine were injected into each group. We obtained blood samples 1 day before the first injection and 1 week after each of the three subsequent injections. We analyzed the serum by ELISA to identify IgG antibodies directed against M2e. The anti-M2e titer in HSA/M2e group and vaccine group were higher than that in PBS group (P<0.05). The anti-M2e was arised from the second week after immunization and rased with the increasing of the number of immunization. The titer was highest in the eighth week.In order to estimate the cellular immunity, we got the splenic lymphocyte of mice 7 days after the last immunization to do experiment of detection of T lymphocyte subtype, lymphocyte transformation and IFN-γ. The results of flow cytometric analysis showed that cell population of CD4+ and CD8+ T lymphocyte of recombination protein group and influenza vaccine group were higher than that of PBS group, but the cellular immunity of influenza vaccine group was excellent especially. The results of lymphocyte transformation demonstrated that the SI index of recombination protein group was higher than that of PBS group , but lower than that of influenza vaccine group (P<0.01) when stimulated with ConA and antigen. The results showed that the numbers of IFN-γ-secreting cell of all experimental groups are more than that of PBS control group evidently (P<0.05), and the numbers of IFN-γ-secreting cell of influenza vaccine group are higher than that of recombination protein . 3.2 study on challenge experiment with influenza A virusMice were infected intranasally (i.n.) with H1N1 and H3N2 virus for about 7 days after the last immunization. Mice were sacrificed 6 days after infection. We got lung of mice and weighed it to calculate the index of lung and inhibitory ratio of the index of lung. The index of lung of PBS group was obviously higher than that of recombination protein group and influenza vaccine group (P<0.05), which were (14.28±1.21) mg/g and (13.89±1.47) mg/g, while recombination protein group and influenza vaccine group were only (11.03±1.08) mg/g, (11.18±1.32) mg/g and (10.45±1.13) mg/g, (10.32±1.40) mg/g, respectively. There was no significant statistical difference between all experimental groups and PBS group (P <0.05).The lung of mice were made to cell suspension and the lung virus titers were determined on MDCK cells. The results showed that the lung virus titers of PBS group were higher than that of recombination protein group and influenza vaccine group, significantly (P<0.05), but he lung virus titers of recombination protein group and influenza vaccine group were no significant difference.Histopathological changes of lung tissues: The lung was taken out and sliced in the normal regulations slice and dyed with HE. The morphological changes of lung tissue was observed under optical microscope. The lung volume of mice of PBS group increased and engorged, while the lungs of normal mice were pink and smooth. About the lung tissues of recombination protein group and influenza vaccine group, the structures of lung alveolus were good and infiltrative phlegmasia cells were less. While many phlegmasia secretion substance and phlegmasia cells infiltrated in lung alveolus of mice of PBS group and the structures of lung alveolus were mostly destroyed.The weight and the number of death of mice were observed 14 days after the virus challenge. The results demonstrated that the weight of PBS group decreased obviously to die after the challenge and all mice of PBS group died after about 10 days .While the weight of recombination protein group and influenza vaccine group decreased in a short time, then recoverd to normal level gradually . The mortality of mice of PBS group were about 87.5% and 100%, while the mortality of recombination protein group were 25%, 37.5% and influenza vaccine group only were 12.5%, respectively. So we consider recombination protein and influenza vaccine could protect the mice against the challenge of the influenza virus.To sum up, in this study we constructed and screened the high-level expression HSA/M2e Pichia pastoris engineering strains, and obtained a high level expression with a yield of 1080 mg/L in a 80 L fermentor by optimizing the main parameters affecting fermentation such as pH, DO and so on.We also established a new method for large-scale fermentation and purification of HSA/M2e in Pichia pastoris. After immunization for three times, the purified HSA/M2e could induce specific cellular immunity and humoral immunity in the BALB/C mice, which could protect the mice against the challenge of lethal dose of H1N1 and H3N2 virus. Our study made the foundation of further exploration on the broad-spectrum influenza vaccine providing cross-protection against different influenza variants.