Expression, processing and high level secretion of a virus toxin in fission yeast (2001)

Heintel T., Zagorc T., and Schmitt MJ.

The virally encoded K28 toxin of Saccharomyces cerevisiae kills sensitive yeast cells in a multi-step receptor-mediated fashion by cell cycle arrest and inhibition of DNA synthesis. In vivo, the toxin is translated as a 38 kDa preprotoxin (pptox) which is enzymatically processed to the biologically active alpha/beta heterodimer during passage through the yeast secretory pathway. Here, we demonstrate that Schizosaccharomyces pombe, a yeast from which no natural toxin-secreting killer strains are known, is perfectly capable of expressing a killer phenotype. Episomal as well as integrating K28 pptox gene cassettes were constructed that allowed a tightly thiamine-regulated killer phenotype expression under transcriptional control of the Sch. pombe nmt1 promotor. Northern analysis of the toxin-coding transcript as well as Western analysis of the secreted toxin indicated that fission yeast is capable of expressing a correctly processed and fully functional virus toxin. Moreover, toxin secretion in recombinant Sch. pombe was at least ten-fold higher than in any natural and/or recombinant Sac. cerevisiae killer strain, indicating that pptox-derived vectors might be attractive in the fast growing field of heterologous protein expression and secretion in yeast.

Appl Microbiol Biotechnol. 56; 165-72.

The fraction of perforin-expressing HIV-specific CD8 T cells is a marker for disease progression in HIV infection (2002)

Heintel T., Sester M., Rodriguez MM., Krieg C., Sester U., Wagner R., Pees HW., Gartner B., Maier R., and Meyerhans A.

OBJECTIVE: Perforin is an important component of the death machinery of cytotoxic T cells (CTL). To evaluate functional differences between HIV- and cytomegalovirus (CMV)-specific CTL of coinfected patients, the frequencies of the respective perforin-expressing T cells were analysed in a rapid whole blood assay. METHODS: Whole blood of HIV- and CMV-infected individuals was specifically stimulated by HIV-1 Pr55(gag) or complete CMV antigen, and activation-induced intracellular cytokine and perforin expression in CD8 T cells was analysed by flow cytometry. RESULTS: Perforin-expressing HIV-1- and CMV-specific CD8 T cells can be quantified simultaneously. Within a patient, the frequency of such HIV-specific CD8 T cells in peripheral blood was lower than the frequency of the respective CMV-specific cells. The number of the perforin-expressing HIV-specific CD8 T cells inversely correlated with the peripheral blood CD4 T cell count. CONCLUSIONS: The differential fractions of perforin-expressing virus-specific CD8 T cells in HIV and CMV double infection might be caused by differences in priming and trafficking to or from replication sites. However, without knowing the underlying mechanism, the fraction of perforin-expressing HIV-specific CD8 T cells provides another surrogate marker for disease progression.

AIDS 26, 1497-501.

Specific activation of CMV-primed human T lymphocytes by cytomegalovirus pp65 expressed in fission yeast (2003)

Breinig, F.*, Heintel, T.*, Schumacher, A., Meyerhans, A., and Schmitt, M. J.

Threatening virus infections constantly illustrate the growing need for novel vaccines that specifically induce efficient T cell-mediated immune responses. In this study, we used a human whole blood assay to determine the activation of antigen-specific human T lymphocytes by a viral antigen of human cytomegalovirus (HCMV). The major HCMV tegument protein pp65, recombinantly expressed in fission yeast (Schizosaccharomyces pombe), specifically activated antigen-specific CD4- and CD8-positive memory T cells in blood of HCMV seropositive donors. Moreover, the immune response against recombinant pp65, in particular that of CD8 class I MHC restricted cytotoxic T cells, was similar to the response against the intact HCM virus. Since fission yeast cells per se did not activate a significant number of human T lymphocytes ex vivo, the system described here might represent a novel approach in vaccine development as well as in the identification of vaccine candidates directly from human whole blood.

FEMS Immunol Med Microbiol 38; 231-239. * Equally contributing coauthors.

Extensive CD8 T lymphocyte responses against various yeast genera in humans (2003)

Heintel, T.*, Breinig, F.*, Schmitt, M. J., and Meyerhans, A.

The human cellular immune response against 14 distantly related yeast species was analyzed by intracellular cytokine staining of lymphocytes after ex vivo stimulation of whole blood. While the CD4 T cell response was marginal, extensive MHC class I-restricted CD8 T cell responses were detected against a number of species including spoiling, environmental and human pathogenic yeasts. The yeast-specific CD8 T cells expressed interferon-g but lacked expression of CD27 and CCR7, indicating that they were end-differentiated effector memory cells. Mainly intact yeast cells rather than spheroplasts were able to induce cytokine expression in T cells demonstrating that the dominant immunogens were located in the yeast cell wall. Together these data underline the importance of the cellular immune response in protecting humans against yeast and fungal infections. And, from another perspective, recombinant yeast suggests itself as a potential vaccine candidate to efficiently induce antigen-specific CD8 T cell responses.

FEMS Immunol Med Microbiol 39:279-286. * Equally contributing coauthors.

Viral preprotoxin signal sequence allows efficient GFP secretion in Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe (2004)

Eiden-Plach, A., Zagorc, T., Heintel, T., Carius, Y., Breinig, F., and Schmitt, M. J.

Besides its importance as model organism in eukaryotic cell biology, yeast also developed into an attractive host for the expression, processing and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae and Schizosaccharomyces pombe) by using the green fluorescent protein (GFP) as reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1, TPI1) or from inducible and/or repressible yeast promoters (GAL1, AOX1, NMT1). In each case, GFP entered the secretory pathway of the corresponding host cell, and confocal fluorescence microscopy as well as SDS-PAGE and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.

Appl Environ Microbiol 70; 961-966.

Foreign proteins targeted to successful secretion in yeast species (2004)

Schmitt, M. J. et al.

No abstract available.

ASM News 70; 185.

Ein virales Killertoxin-Sekretionssignal zur effizienten Proteinsekretion (2004)

Breinig, F., Eiden-Plach, A., Breinig, T., and Schmitt, M. J.

Hefe ist zu einem attraktiven Produktionsorganismus in der Biotechnologie geworden, um rekombinante Proteine zu exprimieren, prozessieren und sekretieren. Obgleich die meisten heterologen Proteine im Zytosol exprimiert werden, blieb deren erfolgreiche Sekretion in das Hefe-Kulturmedium bisher auf wenige erfolgreiche Fälle beschränkt. Vor kurzem konnten wir zeigen, daß eine vom K28-Präprotoxin abgeleitete virale Aminosäure-Sequenz als neuartiges hocheffizientes Sekretionssignal für Fremdproteine dienen kann, auch bei den mit Saccharomyces weniger verwandten, biotechnologisch relevanten Hefe-Species. Die virale Präprotoxin-Signalsequenz kann möglicherweise als neuartiges Werkzeug eingesetzt werden, um eine effiziente Proteinsekretion sicherzustellen. Derzeit fokussieren wir daher unsere Arbeit auf die Toxin-getriebene Sekretion pharmazeutisch und biotechnologisch bedeutender Proteine und Biopharmazeutika in verschiedenen Hefe-Arten.

Laborwelt 5: 4-7.

Forms and Function of Intracellular HIV DNA (2004)

Meyerhans A, Breinig T., Vartanian J-P., and Wain-Hobson S.

Like all retroviruses HIV DNA exists in a variety of forms. When quantified, the provirus is clearly in a minority. The majority of viral DNA forms are dead-ends. In vivo probably upwards of 100 virions are necessary to infect a typically infected cell. Because of this, there is a strong stochastic component to determining which virions make it to the provirus. These findings help reconcile many basic features of HIV virology with general biology.

In: HIV Sequence Compendium 2003; pp. 14-21. Edited by: Leitner T, Foley B, Hahn B, Marx P, McCutchan F, Mellors J, Wolinsky S, and Korber B. Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, LA-UR number 04-7420.

A genetic algorithm approach to simulating HIV evolution reveals the strong impact of multiply infected cells and recombination (2005)

Bocharov, G., Ford, N. J., Edwards, J., Breinig, T., Wain-Hobson, S., and Meyerhans, A.

It has been previously shown that the majority of HIV-1 infected splenocytes can harbor multiple, divergent proviruses with a copy number ranging from 1 to 8 (Jung et al., 2002). This implies that besides point mutations, recombination should be considered as an important mechanism in the evolution of HIV within an infected host. To explore in detail the possible contributions of multi-infection and recombination on HIV evolution, we have simulated in silico the effects of major microscopic parameters of HIV replication (i.e. the point mutation rate, the crossover number, the recombination rate, the provirus copy number) on macroscopic characteristics (such as the Hamming distance, the abundance of n-point mutants). Simulations predict that multiple provirus copies per infected cell and recombination act in synergy to speed up the development of sequence diversity. Point mutations can be fixed for some time without fitness selection. The time needed for the selection of multiple mutations with increased fitness is highly variable, supporting the view that stochastic processes may contribute substantially to the kinetics of HIV variation in vivo.

J Gen Virol. 86, 3109-18.

Antigen-specific T cell responses: Determination of their frequencies, homing properties, and effector functions in human whole blood (2006)

Breinig, T., Sester, M., Sester, U., and Meyerhans, A.

Several prevalent and life-threatening agents enter the organism via the mucosa. In this case, a mucosal cellular immune response is essential for protection and is therefore considered the main objective of vaccination. The frequency of antigen-specific CD4(+) and CD8(+) T cells can be determined directly in human whole blood by a combination of surface marker and intracellular cytokine staining. Immune cells primed in the mucosal compartment also migrate through the blood and can be identified by expression of the gut-specific homing receptor alpha4beta7. Simultaneously, these lymphocytes can be functionally characterized regarding their differentiation status by analysis of CD45RO and CD27 expression and effector functions by measuring intracellular perforin or granzyme B content. Thus, the technique described in the paper is a powerful tool for clinical monitoring of the total cellular immune response to complex antigens during infection or vaccination.

Methods 38, 77-83.

Cross-presentation of HLA class I epitopes from influenza matrix protein produced in Saccharomyces cerevisiae (2006).

Wadle, A.,Held, G., Neumann, F., Kleber, S., Wuellner, B., Asemissen, AM., Kubuschok, B., Scheibenbogen, C., Breinig, T., Meyerhans, A., and Renner, C.

Here we report that genetically engineered yeast of the strain Saccharomyces cerevisiae expressing full-length influenza matrix protein (IMP) attached to the yeast cell wall are a very versatile host for antigen delivery. Feeding of dendritic cells with either intact yeast expressing IMP protein or soluble IMP protein cleaved off the cell wall resulted in protein uptake, processing and cross-presentation of IMP-derived peptides. This process was analysed using previously established T-cell lines recognizing the immuno-dominant 58–66 peptide when presented by HLA-A2*0201 complexes. In addition, IMP_58–66/HLA-A2*0201-specific antibodies were selected from a naive phage library which confirmed that peptide presentation was an active process of endocellular uptake and not just a result of external peptide loading. Moreover, MHC peptide antibodies could block the recognition of peptide-presenting dendritic cells by IMP_58–66-specific T-cells in a dose dependent manner. There was no difference in T-cell recognition when either intact yeast or yeast cell extracts were used for DC feeding. Together, these data demonstrate that yeast derived proteins either in their soluble form or as part of a whole yeast vaccine are taken up, processed and presented by dendritic cells in HLA class I context.

Vaccine 24, 6272-81.

The Autographa californica nuclear polyhedrosis virus AcNPV induces functional maturation of human primary monocyte-derived dendritic cells. (2006)

Schütz, A., Scheller, N., Breinig, T., and Meyerhans, A.

The initiation of an adaptive immune response is critically dependent on the activation of dendritic cells (DCs). Therefore, vaccination strategies targeting DCs have to ensure a proper presentation of the immunogen as well as an activation of DCs to accomplish their full maturation. Viral vectors can achieve gene delivery and a subsequent presentation of the expressed immunogen, however, the immunization efficiency may be hampered by an inhibition of DC activation. Here we report that the insect born Autographa californica nuclear polyhedrosis virus (AcNPV), which is already used for genetic immunization, is able to activate human monocyte-derived DCs. This activation induces the production of tumor necrosis factor alpha (TNF-alpha), an up-regulation of the surface molecules CD83, CD80, CD86, HLA-DR and HLA-I and increases the T cell stimulatory capacity of DCs. Thus, AcNPV represents a promising vector for vaccine trials.

Vaccine 24, 7190-96.

Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression. (2007)

Powilleit, F., Breinig, T., and Schmitt, M.J.

A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae) dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs) as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i) a heterologous model protein (GFP), (ii) a per se toxic protein (K28 a-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

PLoS One 2, e415.

No abstract available yet.

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