Influenza Vaccine Study In α-1, 6-mannosyltransferases Defective Pichia Pastoris

Many envelope proteins of virus were glycoproteins, such as the hemagglutinin and neuraminidase of influenza, the preS of hepatitis B virus, the gp120 of human immunodeficiency virus, and so on. Most of these glycoproteins were protective antigens of anti-virus vaccines. Current researches about vaccines against these antigens were mainly focused on the antigen epitopes of proteins or polysaccharides, and seldom focused on the role of glycans played on glycoprotein vaccines. Many antigen presenting cells contained mannose receptor, so, the mannose might participate in the process of antigen presenting. Lam et al. found that ovalbumin expressed by pichia pastoris was mannose-glycosylated, and the immunogenicity of this ovalbumin was stronger than the non-glycosylated ovalbumin expressed by E.coli [1]. This indicated that mannose-glycosylation might enhance the immunogenicity of antigen.Most of currently licensed influenza vaccines were produced in embryonated hen's eggs. Considering the time-consuming and other disadvantages of the use of eggs as the substrate for influenza vaccine, developing genetic engineering vaccine was one of the most important orientation in influenza vaccine research. The glycoprotein neuraminidase was one of the major antigens of influenza vaccine, and developing neuraminidase as influenza vaccine was part of influenza vaccine research. Yeast was one of the advantageous vaccine production host systems, and had been successful used in the production of non-glycoprotein vaccines, such as HBV and HPV vaccines. However, Martinet et al. had found that N2-type glycoprotein neuraminidase expressed in wild-type Pichia pastoris GS115 was hyper-glycosylated and contained up to 30-40 mannose residues. This glycan chain was quite different from that of native neuraminidase in virus. Using this hyper-glycosylated neuraminidase as immunogen, it could only protect 50% mice against lethal influenza, and Martinet et al. also found that recombinant neuraminidase with decreased glycosylation modification by site-specific mutagenesis was somewhat better to elicit an immune response than wild-type neuraminidase [2]. However, it was unknown whether this site-specific mutagenesis and the reduction of glycosylation sites might lead to the misfold of the protein and the change of antigen epitope. Therefore, this study was focused on how the immunogenicity of neuraminidase was affected when decreasing the size of glycan chains of neuraminidase through blocking the hyper-glycosylation synthesis pathway of neuraminidase expressed in yeast, instead of reducing the site number of glycosylation in neuraminidase. So, theα-1,6-mannosyltransferases (OCH1) gene defective Pichia pastoris was used for neuraminidase production. This OCH1 defection blocked the hyper-glycosylated N-glycan synthesis in Pichia pastoris, and stopped the modification in low-glycosylation form.Thus, to investigate the impact of different glycosylation modification on the immunogenicity of influenza neuraminidases, three different host strains of wild-type Pichia pastoris, the OCH1 defective Pichia pastoris and E.coli were used.The yeast expression vector pPIC9-NA and the E.coli expression vector pET22b-NA were constructed respectively. Then, they were used to transform the wild-type Pichia pastoris, the OCH1 defective Pichia pastoris and E.coli BL21 (DE3). Eventually, three different recombinant strains which contained the neuraminidase gene were gained.After recombinant strains cultivation and proteins purification, purified neuraminidases were harvested. The molecular weight of neuraminidases produced by wild-type Pichia pastoris, OCH1 defective Pichia pastoris and E.coli were 75kDa, 55kDa and 45kDa in SDS-PAGE analysis. After treatment with PNGaseF, the molecular weight of all neuraminidases decreased to 45kDa which was in agreement with the theoretical mass calculated from the predicted amino acid sequence. So, the oligosaccharides of neuraminidases produced in wild-type Pichia pastoris, OCH1 defective Pichia pastoris and E.coli were 40% (hyper-glycosylated), 18% (low-glycosylated) and 0% (non-glycosylated), respectively.BALB/c mice were immunized with these neuraminidases. To assay the titers of nature neuraminidase-special IgG elicited by different neuraminidases, a commercial vaccine containing N1-type neuraminidase which produced in embryonated hen's eggs was used as coated antigen. In 1μg/mouse groups, only low-glycosylated neuraminidase could elicit high neuraminidase special antibody titer, it was 1:5500, while, it was only 1:10 (P<0.01) and 1:13 (P<0.01) elicited by hyper-glycosylated neuraminidase and non-glycosylated neuraminidase respectively. Even after third immunization, antibody titer elicited by hyper-glycosylated neuraminidase was 1:20 (P<0.05) and antibody titer elicited by non-glycosylated neuraminidase increased slightly, it was 1:630. After third immunization, antibody titer elicited by low-glycosylated neuraminidase was similar to the titer after second immunization, it was 1:4700.On the basis of the results mentioned above, the relationship between the injection dose of neuraminidases and the elicited titers of nature neuraminidase-special IgG was further studied.After second immunization, in low-glycosylated neuraminidase group, special antibody titers increased from 1:70 to 1:5500 with the dose of neuraminidase increasing from 0.2μg to 1μg, but in hyper-glycosylated neuraminidase group, special antibody titers were from 1:20 to 1:10 when the dose of neuraminidase increasing from 0.2μg to 1μg, high antibody titer could only be found when the injection dose increased to 3μg, it was 1:6500. The change of antibody titer elicited by non-glycosylated neuraminidase with different injection dose was similar to that of hyper-glycosylated neuraminidase.After third immunization, in 0.2μg/mouse injection dose group, low-glycosylated neuraminidase could elicit 1:4900 special antibody titer, while, high-glycosylated neuraminidase could only elicit 1:10 special antibody titer. The change of antibody titer elicited by non-glycosylated neuraminidase with different injection dose was similar to that of hyper-glycosylated neuraminidase, only when injection dose increased to 3μg/mouse, they could elicit high special antibody titers. When the injection dose increased to 3μg/mouse, all of these neuraminidases could induce high antibody titers, they were 1:36000 by hyper-glycosylated neuraminidase, 1:25000 by low-glycosylated neuraminidase and 1:7000 by non-glycosylated neuraminidase.These results demonstrated that different from hyper-glycosylated neuraminidase produced by wild-type Pichia pastoris, neuraminidase produced by OCH1 defective Pichia pastoris was present in a low-glycosylated modification form. Compared with hyper-glycosylated or non-glycosylated neuraminidase, low-glycosylated neuraminidase was better to elicit high specical antibody against nature neuraminidase. This indicated that low-glycosylated neuraminidase was hopeful when exploited as candidated genetic engineering influenza vaccine, and the OCH1 defective Pichia pastoris GJK01 which had low-glycosylation modification was a promising system for the research and production of glycoprotein vaccine.