Wolfgang A. Renner

Inverse metabolic engineering: A strategy for directed genetic engineering of useful phenotypes

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counter-balancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration.

A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses

Virus like particles (VLPs) are known to induce potent B cell responses in the absence of adjuvants. Moreover, epitope-specific antibody responses may be induced by VLPs that contain peptides inserted in their immunodominant regions. However, due to steric problems, the size of the peptides capable of being incorporated into VLPs while still permitting capsid assembly, is rather limited. While peptides genetically fused to either the N- or C-terminus of VLPs present fewer assembly problems, the immune responses obtained against such epitopes are often limited, most likely because the epitopes are not optimally exposed. In addition, such particles may be less stable in vivo. Here, we show that peptides and proteins engineered to contain a free cys can be chemically coupled to VLPs formed from the hepatitis B core antigen (HBcAg) containing a lys in the immuno-dominant region. By using this approach steric hindrance of capsid assembly is abrogated. Peptides or protein coupled to VLPs in an oriented fashion are shown to induce strong and protective B cell responses even against self-epitopes in the absence of adjuvants. This molecular assembly system may be used to induce strong B cell responses against most antigens.

Nonmethylated CG Motifs Packaged into Virus-Like Particles Induce Protective Cytotoxic T Cell Responses in the Absence of Systemic Side Effects

DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags. CpGs are therefore among the most promising adjuvants known to date. Nevertheless, CpGs are characterized by two drawbacks. They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly. We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qβ is a simple and attractive method to reduce these two problems. CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability. In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity. In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8+ T cells (4–14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors. Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

A therapeutic vaccine for nicotine dependence: preclinical efficacy, and phase I safety and immunogenicity

Nicotine is the principal addictive component in tobacco, and following uptake acts in the central nervous system. The smoking‐cessation efforts of most smokers fail because a single slip often delivers sufficient nicotine to the brain to reinstate the drug‐seeking behaviour. Blocking nicotine from entering the brain by induction of specific antibodies may be an effective means to prevent such relapses. The hapten nicotine was coupled to virus‐like particles (VLP) formed by the coat protein of the bacteriophage Qb. In preclinical experiments, this Nicotine–Qb VLP (NicQb) vaccine induced strong antibody responses. After intravenous nicotine challenge, vaccinated mice exhibited strongly reduced nicotine levels in the brain compared with control mice. In a phase I study, 32 healthy non‐smokers were immunized with NicQb. The vaccine was safe and well‐tolerated. All volunteers who received NicQb showed nicotine‐specific IgM antibodies at day 7 and nicotine‐specific IgG antibodies at day 14. Antibody levels could be boosted by a second injection or the addition of Alum as an adjuvant and the antibodies had a high affinity for nicotine. These data suggest that antibodies induced by NicQb may prevent relapses by sequestering nicotine in the blood of immunized smokers.

A temperature-regulated replicon-based DNA expression system

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Assessment of clinical efficacy of CYT003-QbG10 in patients with allergic rhinoconjunctivitis: a phase IIb study.

Background Allergic symptoms are generally caused by exposure to substances to which people have become sensitized. Associated with this is an ‘unbalanced’ Th1/Th2 immune response with T cell responses skewed towards the production of Th2 cytokines, IL‐4, 5, and 13 and high levels of IgE antibodies. Current immune modulating therapies require the use of allergens, carrying the risk to induce potentially severe allergic reactions.

Virus-like particles as a modular system for novel vaccines

Induction of protective immune responses with recombinant antigens is a major challenge for the vaccine industry. Here we present a molecular assembly system that renders antigens of choice highly repetitive. Using this method, efficient antibody responses may be induced in the absence of adjuvants resulting in protection from viral infection and allergic reactions.

The Growth Factor Inhibitor Suramin Reduces Apoptosis and Cell Aggregation in Protein-Free CHO Cell Batch Cultures

We have previously shown that Chinese hamster ovary (CHO) cells capable of growing in medium free of exogenous proteins die by apoptosis during all stages of a batch culture (Zanghi et al., 1999 ). On the basis of the hypothesis that extracellular death factors might be important in apoptosis under these conditions, we examined the effect of the growth factor inhibitor and antitumor agent suramin on CHO cell growth and apoptosis in serum‐free culture. Suramin protected against apoptosis during exponential growth, as indicated by the absence of DNA laddering and an increase in cell viability from roughly 70% to above 95%. Suramin also effectively dispersed cell aggregates so that single‐cell suspension culture was possible. However, suramin did not protect against apoptosis during the death phase, in contrast to serum, suggesting that antiapoptotic factors in the serum remain to be discovered. The increased viable cell yield following suramin supplementation resulted in a 40% increase in product yield, based on results with cells expressing recombinant secreted alkaline phosphatase. Polysulfated compounds dextran sulfate and polyvinyl sulfate worked nearly as well as suramin in dispersing cell clumps and increasing viable cell yield, which implies that suramins high sulfate group density may be responsible for its effects in cell culture. In addition, suramin was beneficial for long‐term adaptation of CHO cells to protein‐free media suspension culture, and the compound was synergistic with insulin in accelerating this adaptation time.

Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction

Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for >1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.

Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium.

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F‐1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin‐resistance selection pressure all expressed significantly higher amounts of E2F‐1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F‐1 in these cells. CHO K1:E2F‐1 cells are able to proliferate on well‐defined serum‐ and protein‐free basal medium and exhibit an S‐phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two‐dimensional electrophoresis of the intracellular proteins of E2F‐1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F‐1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F‐1 in human fibroblasts. Overexpression of cloned E2F‐1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus‐protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation.