Jana Rödig

Toward Animal Cell Culture–Based Influenza Vaccine Design: Viral Hemagglutinin N-Glycosylation Markedly Impacts Immunogenicity

The glycoproteins hemagglutinin (HA) and neuraminidase are the major determinants of host range and tissue tropism of the influenza virus. HA is the most abundant protein in the virus particle membrane and represents the basis of most influenza vaccines. It has been reported that influenza virus HA N-glycosylation markedly depends on the host cell line used for virus production. However, little is known about how differential glycosylation affects immunogenicity of the viral proteins. This is of importance for virus propagation in chicken eggs as well as for innovative influenza vaccine production in mammalian cell lines. In this study, we investigated the impact of the differential N-glycosylation patterns of two influenza A virus PR/8/34 (H1N1) variants on immunogenicity. Madin–Darby canine kidney cell–derived and Vero cell–derived glycovariants were analyzed for immunogenicity in a TCR-HA transgenic mouse model. Next-generation pyrosequencing validated the congruence of the potential HA N-glycosylation sites as well as the presence of the HA peptide recognized by the TCR-HA transgenic T cells. We show that differential HA N-glycosylation markedly affected T cell activation and cytokine production in vitro and moderately influenced IL-2 production in vivo. Cocultivation assays indicated that the difference in immunogenicity was mediated by CD11c+ dendritic cells. Native virus deglycosylation by endo- and exoglycosidases dramatically reduced cytokine production by splenocytes in vitro and markedly decreased HA-specific Ab production in vivo. In conclusion, this study indicates a crucial importance of HA N-glycosylation for immunogenicity. Our findings have implications for cell line–based influenza vaccine design.

Impact of cultivation conditions on N-glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition).

Vaccine production: upstream processing with adherent or suspension cell lines.

The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered.

Impact of Influenza Virus Adaptation Status on HA N-Glycosylation Patterns in Cell Culture-Based Vaccine Production

The highly abundant and strongly immunogenic influenza envelope glycoprotein hemagglutinin (HA) represents the main component of influenza vaccines. Human influenza vaccines are typically produced in embryonated chicken eggs. In addition, cell culture-derived vaccine production systems are currently being established. Since characteristics of glycoproteins such as the HA can be significantly influenced by N-glycosylation, the impact of host cells considered for vaccine manufacturing needs to be addressed. In this study MDCK cell-derived influenza A/PR/8/34 (H1N1) virus was adapted over four passages in AGE1.CR.pIX-cells. HA N-glycosylation patterns (normalized capillary electropherograms) were determined and analyzed using capillary gel electrophoresis with laser-induced fluorescence detection (each peak represents at least one distinct N-glycan structure). During the adaptation to AGE1.CR.pIX-cells, virus titers 24 hours postinfection improved. HA N-glycosylation patterns of MDCK and AGE1.CR.pIX-derived virus particles differed significantly after the first adaptation step. This clearly suggests that the structure of the viral antigens is strongly influenced by the host cell. Furthermore, AGE1.CR.pIX-derived antigens showed a tendency toward small glycans. Differences between glycan patterns of the four successive passages in AGE1.CR.pIX cell were minor; only low variability in relative peak height was noted in the HA N-glycosylation pattern.

Glycosylation matters : role of viral hemagglutinin N-glycosylation in immunogenicity

Background: Polyvalent vaccination represents a recent attempt to improve the effectiveness of lung cancer immunotherapy. This study aimed to investigate whether a gene expression pattern of tumor-associated antigens (TAA) would exist indicating that their use will be most appropriate for the polyvalent vaccination of Caucasian non-small cell lung carcinoma (NSCLC) patients. We examined the concomitant expression of genes belonging to different TAA families for which expression frequencies either have never been detected in NSCLC or vary widely in the literature. Methods: Tumor material from 23 patients with NSCLC (12 adenocarcinomas, 8 squamous cell carcinomas, 3 bronchoalveolar carcinomas) was examined. mRNA transcripts were detected for 5 genes of the survivin family, 5 MAGE-A genes as well as the genes of human telomerase reverse transcriptase (hTERT) and p53, by the use of quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) or semiquantitative RT-PCR. Results: 15/23 (65%) and 8/23 (35%) tumor samples were found expressing 6-11 and 2-5 out of the 12 examined TAAs, respectively, at levels >1% of the testis reference sample. The most prevalent TAA patterns observed were those of survivin standard (survivinstd)/survivin-2B expressed by 22/23 (95.5%) tumor samples and of survivin-std/survivin-2B/hTERT expressed by 19/23 (82.5%) tumor samples. The expression levels of the survivin-std gene strongly positively correlated to those of the survivin-2B (p=0.001) and the hTERT genes (p=0.031). The number of concomitantly expressed genes was found to be positively correlated to the age of the patients (p=0.001) and the tumor size (p=0.048). Conclusion: This study provides evidence that, in Caucasian patients with NSCLC, highly prevalent expression patterns of TAA genes, predominantly of overexpressed TAAs, do exist. This result implies that the combined use of these TAA could help in designing more effective NSCLC immunotherapeutic protocols.Purpose/Objective: Rheumatoid arthritis (RA) is a chronic autoimmune disease that leads to joint destruction. The recruitment of effectors cells, including monocytes to the joint space is an important step in RA progression and is mediated by chemokines (Ch) and their receptors (ChR). MicroRNAs are a recently discovered class of posttranscriptional regulators. Many members of the miR family are implicated in the regulation of cell movement and migration. Our previous study showed miR-155 is upregulated in RA synovial fluid (SF) monocytes suggesting that this miR may be involved in activation of these cells, including their migration into joint space. We hypothesized that miR-155 could regulates migration of monocytes in RA by modulating the expression of the chemokine and chemokine receptor system. Materials and methods: Peripheral blood (PB) CD14+ cells from healthy controls (HC) and RA patients were transfected with miR-155 mimic or scramble mimic using N-TER nanoparticles and cultured for 48 h. TaQman Low Density Array and multiplex assay was used to evaluate ChR expression and Ch production, respectively. Similar analysis was carried out on bone marrow monocytes (BMM) from miR-155-/- and WT mice. In addition, absolute copy numbers of miR- 155 transcripts in PB and SF CD14+ of RA and HC were assessed by QPCR. Results: PB and SF monocytes in RA patients showed higher copy number of miR-155 compared to HC. Overexpression of miR-155 in HC and RA monocytes did not affect the production of CCL2, CCL7, CCL21, CXCL5, CXCL8, CXCL7, CXCL10 and CX3CL1. In contrast, overexpression of miR-155 induced the production of chemokines such as CCL4, CCL5 and CCL22 in RA monocytes and CCL3 in both RA and HC. Analysis of chemokine receptors in BMM of miR-155-/- and WT mice revealed significantly higher levels of CCR1, CCR2, CCR5 and CXCR4 in miR-155 deficient cells suggesting that miR-155 can act as a negative regulator of these receptors in homeostatic state. As expected, TLR-4 ligand significantly suppressed expression of these receptors in both WT and miR-155-/- cells. Analysis of 3’UTRs of Ch/ ChR (TargetScan) suggests that miR-155 is likely interfering with signaling pathways implicated in Ch/ChR system expression. Conclusions: Deregulation of miR-155 in RA monocytes can contribute to the production of pro-inflammatory chemokines by these cells and to their accumulation at sites of inflammation.Purpose/Objective: Sphingosine kinase (SPHKs), SphK1 and SphK2, have been identified to phosphorylate sphingosine into sphingosine-1- phosphate (S1P). They are involved in a wide variety of cellular responses. S1P acts via S1P Receptors, S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, all of which can be bound and activated specifically by S1P. A defect either in S1P signalling or S1PRs has been associated with many pathologies. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by high levels of proinflammatory cytokine production. Elevated SPHK1, S1P, and S1P1 have been reported in RA synovium. S1P signalling via S1P1 promotes synoviocyte proliferation, increases COX-2 expression and prostaglandin E2 production. This study comprehensively evaluated expression of SPHK1/2 and S1PRs in RA patients compare to healthy controls (HC) and osteoarthritis (OA) in peripheral blood (PB) and synovial tissues, respectively. Materials and methods: mRNA and protein expression of SPHK1/2 and SIPRs were examined in neutrophils, monocytes and T lymphocytes of peripheral blood of 10 HC and RA patients, who met the diagnostic criteria of 2010 ARC / EULAR by QPCR and FACS, respectively. Competitive ELISA assessed SIP in serum of RA patients with remission and relapse and HC. We also performed SPHK 1/2 and SIPRs immunohistochemistry in synovial tissue from 4 RA/ OA patients. Results: S1P was three times high in RA than those observed in HC, also was statistically higher in RA patient with relapse than remission. Intracellular expression of hSPHK1 in RA patients, with opposed to HC, was up regulated 1.4-folds in monocytes and T- lymphocytes with significance expression in CD4T cells. hS1P1 and hS1P3 exhibited a similar expression were up-regulated in neutrophils, while, hS1P5 was statistical high in T cells. In contrast, hS1P4 was down regulated in all sorted cells particularly in CD4T cells. As opposed to OA synovial tissue, RA synovial tissues were strongly positive for hSPHK1 and hS1P1, 3 expressions. Quantitative analysis showed, SPHK1 and hS1P3 are expressed in lining, sub lining and vascular endothelial layer, while hS1P1 expressed mainly in lining and sub lining layers of the RA synovial tissue compared with OA. Conclusions: These results suggest that SPHKs/S1P and its S1PRs might play a role in RA pathogenesis. The clinical significance of S1P as a biomarker for disease activity deserves further attention.