Combating viral contaminants in CHO cells by engineering innate immunity.

Viral contamination in biopharmaceutical manufacturing can lead to shortages in the supply of critical therapeutics. To facilitate the protection of bioprocesses, we explored the basis for the susceptibility of CHO cells to RNA virus infection. Upon infection with certain ssRNA and dsRNA viruses, CHO cells fail to generate a significant interferon (IFN) response. Nonetheless, the downstream machinery for generating IFN responses and its antiviral activity is intact in these cells: treatment of cells with exogenously-added type I IFN or poly I:C prior to infection limited the cytopathic effect from Vesicular stomatitis virus (VSV), Encephalomyocarditis virus (EMCV), and Reovirus-3 virus (Reo-3) in a STAT1-dependent manner. To harness the intrinsic antiviral mechanism, we used RNA-Seq to identify two upstream repressors of STAT1: Gfi1 and Trim24. By knocking out these genes, the engineered CHO cells exhibited activation of cellular immune responses and increased resistance to the RNA viruses tested. Thus, omics-guided engineering of mammalian cell culture can be deployed to increase safety in biotherapeutic protein production among many other biomedical applications.

Antigen-specific induced T regulatory cells impair dendritic cell function via an IL-10/MARCH1-dependent mechanism.

Foxp3(+) T regulatory cells (Tregs) are critically important for the maintenance of immunological tolerance, immune homeostasis, and prevention of autoimmunity. Dendritic cells (DCs) are one of the major targets of Treg-mediated suppression. Some studies have suggested that Treg-mediated suppression of DC function is mediated by the interaction of CTLA-4 on Tregs with CD80/CD86 on the DCs resulting in downregulation of CD80/CD86 expression and a decrease in costimulation. We have re-examined the effects of Tregs on mouse DC function in a model in which Ag-specific, induced Tregs (iTregs) are cocultured with DCs in the absence of T effector cells. iTreg-treated DCs are markedly defective in their capacity to activate naive T cells. iTregs from CTLA-4-deficient mice failed to induce downregulation of CD80/CD86, but DCs treated with CTLA-4-deficient iTregs still exhibited impaired capacity to activate naive T cells. The iTreg-induced defect in DC function could be completely reversed by anti-IL-10, and IL-10-deficient iTregs failed to downregulate DC function. iTreg-treated DCs expressed high levels of MARCH1, an E3 ubiquitin ligase, recently found to degrade CD86 and MHC class II on the DCs and expressed lower levels of CD83, a molecule involved in neutralizing the function of MARCH1. Both the enhanced expression of MARCH1 and the decreased expression of CD83 were mediated by IL-10 produced by the iTregs. Taken together, these studies demonstrate that a major suppressive mechanism of DC function by iTregs is secondary to the effects of IL-10 on MARCH1 and CD83 expression.

A novel tetrameric gp350 1-470 as a potential Epstein-Barr virus vaccine.

Infectious mononucleosis and B-cell transformation in response to infection with Epstein-Barr virus (EBV) is dependent upon binding of the EBV envelope glycoprotein gp350 to CD21 on B-cells. Gp350-specific antibody comprises most of the EBV neutralizing activity in the serum of infected patients, making this protein a promising target antigen for a prophylactic EBV vaccine. We describe a novel, tetrameric gp350-based vaccine that exhibits markedly enhanced immunogenicity relative to its monomeric counterpart. Plasmid DNA was constructed for synthesis, within transfected CHO cells, of a tetrameric, truncated (a.a. 1-470) gp350 protein (gp350(1-470)). Tetrameric gp350(1-470) induced ≈ 20-fold higher serum titers of gp350(1-470)-specific IgG and >19-fold enhancements in neutralizing titers at the highest dose, and was >25-fold more immunogenic on a per-weight basis than monomeric gp350(1-470). Further, epidermal immunization with plasmid DNA encoding gp350(1-470) tetramer induced 8-fold higher serum titers of gp350(1-470)-specific IgG relative to monomer. Tetrameric gp350(1-470) binding to human CD21 was >24-fold more efficient on a per-weight basis than monomer, but neither tetramer nor monomer mediated polyclonal human B-cell activation. Finally, the introduction of strong, universal tetanus toxoid (TT)-specific CD4+ T-cell epitopes into the tetrameric gp350(1-470) had no effect on the gp350(1-470)-specific IgG response in naïve mice, and resulted in suppressed gp350(1-470)-specific IgG responses in TT-primed mice. Collectively, these data suggest that tetrameric gp350(1-470) is a potentially promising candidate for testing as a prophylactic EBV vaccine, and that protein multimerization, using the approach described herein, is likely to be clinically relevant for enhancing the immunogenicity of other proteins of vaccine interest.

Parameters underlying distinct T cell-dependent polysaccharide-specific IgG responses to an intact gram-positive bacterium versus a soluble conjugate vaccine.

IgG anti-polysaccharide (PS) responses to both intact Streptococcus pneumoniae (Pn) and PS conjugate vaccines are dependent on CD4(+) T cells, B7-dependent costimulation, and CD40-CD40-ligand interactions. Nevertheless, the former response, in contrast to the latter, is mediated by an ICOS-independent, apoptosis-prone, extrafollicular pathway that fails to generate PS-specific memory. We show that pre-existing PS-specific Igs, the bacterial surface or particulation, selective recruitment of B cell subsets, or activation and recruitment of Pn protein-specific CD4(+) T cells do not account for the failure of Pn to generate PS-specific IgG memory. Rather, the data suggest that the critical factor may be the lack of covalent attachment of PS to protein in intact Pn, highlighting the potential importance of the physicochemical relationship of PS capsule with the underlying bacterial structure for in vivo induction of PS-specific Igs.

Intact bacteria inhibit the induction of humoral immune responses to bacterial-derived and heterologous soluble T cell-dependent antigens.

During infections with extracellular bacteria, such as Streptococcus pneumoniae (Pn), the immune system likely encounters bacterial components in soluble form, as well as those associated with the intact bacterium. The potential cross-regulatory effects on humoral immunity in response to these two forms of Ag are unknown. We thus investigated the immunologic consequences of coimmunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS) as a model. Coimmunization of mice with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, as well as primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1-microm latex beads in adjuvant. Inhibition was long-lasting and occurred only during the early phase of the immune response, but it was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated Ags are the preferential immunogen for antibacterial Ig responses.

B and CD4+ T-cell expression of TLR2 is critical for optimal induction of a T-cell-dependent humoral immune response to intact Streptococcus pneumoniae.

TLR2(-/-) mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4(+) T-cell-dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T-cell-dependent phosphorylcholine (PC)-specific IgG3 versus the T-cell-independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn-activated TLR2(-/-) BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti-PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4(+)T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2(-/-) mice for a normal CD4(+) T-cell IFN-gamma recall response. Notably, TLR2(-/-) B cells transferred into RAG-2(-/-) mice with WT CD4(+)T cells, or TLR2(-/-) CD4(+)T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti-PC response relative to WT cells. These data are the first to demonstrate a major role for B-cell and CD4(+) T-cell expression of TLR2 for eliciting an anti-bacterial humoral immune response.

Transgenic expression of Bcl-xL or Bcl-2 by murine B cells enhances the in vivo antipolysaccharide, but not antiprotein, response to intact Streptococcus pneumoniae.

IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.

The critical DNA flanking sequences of a CpG oligodeoxynucleotide, but not the 6 base CpG motif, can be replaced with RNA without quantitative or qualitative changes in Toll-like receptor 9-mediated activity.

Double- and single-stranded oligodeoxynucleotides containing unmethylated cytosine-guanosine (CpG) dinucleotides (CpG-ODN) activate immune cells via TLR9. In this report we synthesized hybrid DNA-RNA molecules (HDR) in order to further explore the structure-immune function relationship of CpG-ODN in TLR9 signaling and the potential immunomodulatory properties of RNA. We demonstrate that replacement of the deoxyadenosine flanking sequences, critical for the immune activating properties of CpG-ODN, with a similar number of adenosines, although not guanosines, cytosines, or uracils, maintains complete immunostimulatory activity of the hybrid oligonucleotide in vitro, whereas a similar RNA replacement of even 1 base of the required unmethylated 6 base DNA motif (purine-purine-CpG-pyrimidine-pyrimidine) results in a complete loss of activity. Regardless of whether the critical flanking sequence was RNA or DNA there was no significant change in the quantitative or qualitative immune-stimulating activity, or TLR-specificity of the resulting sequences, thus underscoring the relatively permissive functional role of the flanking sequence, and the more specific role of the motif in mediating TLR9 signaling. These data further support a potential role for RNA in immunomodulation.

Protective role of exogenous polyamines on salinity-stressed rice (Oryza sativa) plants.

Salt-tolerant Pokkali rice plants accumulate higher polyamines (PAs) such as spermidine (Spd) and spermine (Spm) in response to salinity stress, while the sensitive cultivarM-1-48 is unable to maintain high titres of these PAs under similar conditions. The effects of the triamine Spd and the tetramine Spm on physiological and biochemical changes in 12-day-old rice seedlings were investigated during salinity stress to determine whether they could protect the sensitive plants from stress effects. At physiological concentrations Spd and Spm significantly prevented the leakage of electrolytes and amino acids from roots and shoots induced by salinity stress. To different degrees they also prevented chlorophyll loss, inhibition of photochemical reactions of photosynthesis as well as downregulation of chloroplast-encoded genes like psbA, psbB, psbE and rbcL, indicating a positive correlation between salt tolerance and accumulation of higher PAs in rice. The inhibitory effect of salinity stress and its reversal by exogenous PAs were more pronounced in the salt-sensitiveM-1-48 plants than in the tolerant Pokkali plants.