Characterizing Complex Polysera Produced by Antigen-Specific Immunization through the Use of Affinity-Selected Mimotopes

BACKGROUND: Antigen-based (as opposed to whole organism) vaccines are actively being pursued for numerous indications. Even though different formulations may produce similar levels of total antigen-specific antibody, the composition of the antibody response can be quite distinct resulting in different levels of therapeutic activity. METHODOLOGY/PRINCIPAL FINDINGS: Using plasmid-based immunization against the proto-oncogene HER-2 as a model, we have demonstrated that affinity-selected epitope mimetics (mimotopes) can provide a defined signature of a polyclonal antibody response. Further, using novel computer algorithms that we have developed, these mimotopes can be used to predict epitope targets. CONCLUSIONS/SIGNIFICANCE: By combining our novel strategy with existing methods of epitope prediction based on physical properties of an individual protein, we believe that this method offers a robust method for characterizing the breadth of epitope-specificity within a specific polyserum. This strategy is useful as a tool for monitoring immunity following vaccination and can also be used to define relevant epitopes for the creation of novel vaccines

Investigating the potential of thermophilic species for ethanol production from industrial spent sulfite liquor

Thermophilic microorganisms hold a great potential for bioethanol production on waste biomass, due to their ability to utilize pentoses and hexoses alike. However, to date hardly any data on thermophiles growing directly on industrial substrates like spent sulfite liquor (SSL) are available. This contribution investigates the ability of Thermoanaerobacter species to utilize the main sugars in the used SSL (mannose, glucose and xylose) and the effect of process parameters (pH, temperature and sugar concentration) on their growth. Based on these results the strain T. mathranii was chosen for further studies. The ability of T. mathranii to grow directly on SSL was investigated and the effect of several inhibiting substances on growth was elucidated. Furthermore it was tested whether pretreatment with activated charcoal can increase the fermentability of SSL. The fermentations were evaluated based on yields and specific rates. It could be shown that T. mathranii was able to ferment all sugars in the investigated softwood SSL and fermented diluted, untreated SSL (up to 2.7% (w/w) dry matter). Pretreatment with activated charcoal could slightly reduce the amount of phenols in the substrate and thus facilitate growth and ethanol production on higher SSL concentrations (up to 4.7% (w/v) dry matter). Ethanol yields of 0.29-0.44 Cmmol of ethanol per Cmmol sugar were obtained on untreated and pretreated spent sulfite liquor, respectively. These results on an industrial substrate strengthen the claim that thermophilic microorganisms might be the optimal candidates for forest biorefinery

Generation of PHB from spent sulfite liquor using halophilic microorganisms

Halophilic microorganisms thrive at elevated concentrations of sodium chloride up to saturation and are capable of growing on a wide variety of carbon sources like various organic acids, hexose and also pentose sugars. Hence, the biotechnological application of these microorganisms can cover many aspects, such as the treatment of hypersaline waste streams of different origin. Due to the fact that the high osmotic pressure of hypersaline environments reduces the risk of contamination, the capacity for cost-effective non-sterile cultivation can make extreme halophilic microorganisms potentially valuable organisms for biotechnological applications. In this contribution, the stepwise use of screening approaches, employing design of experiment (DoE) on model media and subsequently using industrial waste as substrate have been implemented to investigate the applicability of halophiles to generate PHB from the industrial waste stream spent sulfite liquor (SSL). The production of PHB on model media as well as dilutions of industrial substrate in a complex medium has been screened for by fluorescence microscopy using Nile Blue staining. Screening was used to investigate the ability of halophilic microorganisms to withstand the inhibiting substances of the waste stream without negatively affecting PHB production. It could be shown that neither single inhibiting substances nor a mixture thereof inhibited growth in the investigated range, hence, leaving the question on the inhibiting mechanisms open. However, it could be demonstrated that some haloarchaea and halophilic bacteria are able to produce PHB when cultivated on 3.3% w/w dry matter spent sulfite liquor, whereas H. halophila was even able to thrive on 6.6% w/w dry matter spent sulfite liquor and still produce PHB

Three-dimensional modeling of putative epitopes.

<p>Three-dimensional model of rat HER-2 is presented in two orientations. <i>A.</i> clusters found for HER-2_FLserum, <i>B.</i> clusters found for HER-2_ECD-serum, <i>C.</i> clusters found for HER-2_TUBO –serum. Serum- specific clusters of amino acid pairs are numbered. Amino acid are coloured according to RasMol amino acid colour code: red = Glu and Asp, blue = Arg and Lys, brown = Pro, orange = Ser and Thr, Cyan = Asn and Gln, violet = Trp and Phe, purple = His.</p

Theoretical prediction of HER-2 epitopes.

*<p>potential calculated for every amino acid using DiscoTope software and characterizing immunogenic property of a given amino acid.</p

Occurrence of statistically significant amino acid pairs within mimotope collections.

a<p>Polyserum used for mimotope isolation.</p>b<p>Single letters amino acid codes (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005309#s3" target="_blank">Methods</a>).</p>c<p>Number of mimotopes carrying this generalized pair within the specific collection.</p>d<p>Unique pairs are presented in bold, pairs shared by two datasets are presented in italics.</p

Clusters predicted by computer algorithm.

<p>Clusters predicted by computer algorithm.</p

Antigen structure influences the composition of the polyclonal response.

<p><i>A.</i> ELISA plate wells were coated with recombinant rat ECD and reacted with serial dilutions of mouse sera. <i>B.</i> Recombinant extracellular domain (ECD) of rat HER-2 was treated (<i>reduced protein</i>) or not (<i>native protein</i>) with β-mercaptoethanol and run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000. <i>C.</i> 10⁶ Tubo cells over-expressing rat HER-2 were reacted with serial dilutions of mouse sera, probed with anti-mouse antibody conjugated with PE and fluorescence intensity was measured by flow cytometry. Each point is reflective of at least 10 000 events. <i>D.</i> Recombinant extracellular domain of rat HER-2 or human HER-2 were run in 10% SDS PAGE, transferred onto nitrocellulose membrane and probed with mouse sera diluted 1∶1000 or anti-HER-2 antibody. <i>E.</i> 500 TUBO cells were plated into 96 well plate in DMEM medium supplemented with 5% FBS and incubated 1–2 weeks in the presence of IgGs purified from mouse sera at a final concentration of 25 µg/ml.. The cell proliferation was measured according to manufacturer instruction by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay. The error bars reflect the mean+/−sem for 4 samples. These data represent the results of pooled serum from 5 vaccinated mice per group. Each experiment was replicated at least 3 times and representative results are shown.</p

Occurrence of statistically significant amino acids within mimotope collections.

a<p>Polyserum used for mimotope isolation.</p>b<p>The frequency was determined as: (# specific residues/total # of residues in the collection) ×100.</p

Mimotope sequences of affinity-selected phage.

a<p>Polyserum used for mimotope isolation.</p