In vitro evolution of myc-tag antibodies: in-depth specificity and affinity analysis of Myc1-9E10 and Hyper-Myc.

One of the most widely used epitope tags is the myc-tag, recognized by the anti-c-Myc hybridoma antibody Myc1-9E10. Combining error-prone PCR, DNA shuffling and phage display, we generated an anti-c-Myc antibody variant (Hyper-Myc) with monovalent affinity improved to 18 nM and thermal stability increased by 37%. Quantification of capillary immunoblots and by flow cytometry demonstrated improved antigen detection by Hyper-Myc. Further, three different species variants of this antibody were generated to allow the use of either anti-human, anti-mouse or anti-rabbit Fc secondary antibodies for detection. We characterized the specificity of both antibodies in depth: individual amino acid exchange mapping demonstrated that the recognized epitope was not changed by the in vitro evolution process. A laser printed array of 29,127 different epitopes representing all human linear B-cell epitopes of the Immune Epitope Database allowing to chart unwanted reactivities with mimotopes showed these to be very low for both antibodies and not increased for Hyper-Myc despite its improved affinity. The very low background reactivity of Hyper-Myc was confirmed by staining of myc-tag transgenic zebrafish whole mounts. Hyper-Myc retains the very high specificity of Myc1-9E10 while allowing myc-tag detection at lower concentrations and with either anti-mouse, anti-rabbit or anti human secondary antibodies.

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity.

Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.

Cancer is not one disease but rather a collection of disorders. As such there are many reasons why someone may develop cancer during their lifetime, including the individual's family history, lifestyle and habits. Infections with certain viruses can also lead to cancer and human papillomaviruses are viruses that establish long-term infections that may result in cancers including cervical and anal cancer, and the most common form of cancer worldwide, non-melanoma skin cancer. The human papillomavirus, or HPV for short, is made up of DNA surrounded by a protective shell, which contains many repeats of a protein called L1. These L1 proteins stick to the surfaces of human cells, allowing the virus to get access inside, where it can replicate before spreading to new cells. The immune system responds strongly to HPV infections by releasing antibodies that latch onto L1 proteins. It was therefore not clear how HPV could establish the long-term infections and cause cancer when it was seeming being recognized by the immune system. Now, Fu et al. have used the Southern multimammate mouse, Mastomys coucha, as a model system for an HPV infection to uncover how papillomaviruses can avoid the immune response. This African rodent is naturally infected with a skin papillomavirus called MnPV which, like its counterpart in humans, can trigger the formation of skin warts and malignant skin tumors. Fu et al. took blood samples from animals that had been infected with the virus over a period of 76 weeks to monitor their immune response overtime. This revealed that, in the early stages of infection, the virus made longer-than-normal versions of the L1 protein. Further analysis showed that these proteins could not form the virus's protective shell but could trigger the animals to produce antibodies against them. Fu et al. went on to show that the antibodies that recognized the longer variants of L1 protein where "non-neutralizing", meaning that could not block the spread of the virus, which is a prerequisite for immunity. It was only after a delay of four months that the animals started making neutralizing antibodies that were directed against the shorter L1 proteins that actually makes up the virus's protective coat. These findings suggest that virus initially uses the longer version of the L1 protein as a decoy to circumvent the attention of the immune system and provide itself with enough time to establish an infection. The findings also have implications for other studies that have sought to assess the success of an immune response during a papillomavirus infection. Specifically, the delayed production of the neutralizing antibodies means that their presence does not necessarily indicate that a patient is not already infected by a papillomavirus that in the future may cause cancer.

Printed peptide arrays identify prognostic TNC serumantibodies in glioblastoma patients.

Liquid biopsies come of age offering unexploited potential to monitor and react to tumor evolution. We developed a cost-effective assay to non-invasively determine the immune status of glioblastoma (GBM) patients. Employing newly developed printed peptide microarrays we assessed the B-cell response against tumor-associated antigens (TAAs) in 214 patients. Firstly, sera of long-term (36+ months, LTS, n=10) and short-term (6-10 months, STS, n=14) surviving patients were screened for prognostic antibodies against 1745 13-mer peptides covering known TAAs (TNC, EGFR, GLEA2, PHF3, FABP5, MAGEA3). Next, survival associations were investigated in two retrospective independent multicenter validation sets (n=61, n=129, all IDH1-wildtype). Reliability of measurements was tested using a second array technology (spotted arrays). LTS/STS screening analyses identified 106 differential antibody responses. Evaluating the Top30 peptides in validation set 1 revealed three prognostic peptides. Prediction of TNC peptide VCEDGFTGPDCAE was confirmed in a second set (p=0.043, HR=0.66 [0.44-0.99]) and was unrelated to TNC protein expression. Median signals of printed arrays correlated with pre-synthesized spotted microarrays (p<0.0002, R=0.33). Multiple survival analysis revealed independence of age, gender, KPI and MGMT status. We present a novel peptide microarray immune assay that identified increased anti-TNC VCEDGFTGPDCAE serum antibody titer as a promising non-invasive biomarker for prolonged survival.

Peptide arrays with a chip.

Today, lithographic methods enable combinatorial synthesis of >50,000 oligonucleotides per cm(2), an advance that has revolutionized the whole field of genomics. A similar development is expected for the field of proteomics, provided that affordable, very high-density peptide arrays are available. However, peptide arrays lag behind oligonucleotide arrays. This is mainly due to the monomer-by-monomer repeated consecutive coupling of 20 different amino acids associated with lithography, which adds up to an excessive number of coupling cycles. A combinatorial synthesis based on electrically charged solid amino acid particles resolves this problem. A computer chip consecutively addresses the different charged particles to a solid support, where, when completed, the whole layer of solid amino acid particles is melted at once. This frees hitherto immobilized amino acids to couple all 20 different amino acids in one single coupling reaction to the support. The method should allow for the translation of entire genomes into a set of overlapping peptides to be used in proteome research.

A novel combinatorial approach to high-density peptide arrays.

Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide-protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4). Solid amino acid microparticles were charged by friction and transferred to defined pixel electrodes onto the chip's surface, where they couple to a functional polymer coating simply upon melting (Fig. 16.1 A-D,F). By applying standard Fmoc chemistry according to Merrifield, peptide array densities of up to 40,000 spots per square centimetre were achieved (Fig. 16.1G). The term "Merrifield synthesis" describes the consecutive linear coupling and deprotecting of L-amino acids modified with base-labile fluorenylmethoxy (Fmoc) groups at the N-terminus and different acid-sensitive protecting groups at their side chains. Removing side chain protecting groups takes place only once at the very end of each synthesis and generates the natural peptide sequence thereby.

The effect of vaccination against porcine circovirus type 2 in pigs suffering from porcine respiratory disease complex.

A field study was conducted to investigate the effect of vaccination against porcine circovirus type 2 (PCV2) in pigs suffering from porcine respiratory disease complex (PRDC). A total of 1542 pigs were allocated randomly into two treatment groups at approximately 20 days of age. Groups received either a Baculovirus-expressed recombinant PCV2 Open Reading Frame (ORF) 2 vaccine or placebo by single intramuscular injection. Median onset of PCV2 viraemia and respiratory signs occurred when animals were 18 weeks old. Vaccination reduced the mean PCV2 viral load by 55-83% (p < 0.0001) and the mean duration of viraemia by 50% (p < 0.0001). During the period of study (from 3 to 25 weeks of age) vaccinated animals exhibited a reduced mortality rate (6.63% vs. 8.67%, difference -2.04%; p = 0.1507), an improved average daily weight gain (649 g/day vs. 667 g/day; difference +18 g/day; p < 0.0001) and a reduced time to market (164.8 days vs. 170.4 days; difference -5.6 days; p < 0.0001). The effects on performance were greatest in the 8-week period between the onset of PCV2 viraemia and the end of finishing. These data demonstrate that vaccination against PCV2 alone can significantly improve the overall growth performance of pigs in a multi-factorial, late occurring disease complex such as PRDC.

Noncontact charge measurement of moving microparticles contacting dielectric surfaces.

In this study examples for a noncontact procedure that allow the description of instant electric charging of moving microparticles that contact dielectric surfaces, for instance, of a flow hose are presented. The described principle is based on the measurement of induced currents in grounded metal wire probes, as moving particles pass close to the probe. The feasibility of the approach was tested with laser printer toner particles of a given size for different basic particle flow and charging conditions. An analytic description for the induced currents was developed and compared to observed effects in order to interpret the results qualitatively. The implementation of the presented procedure can be applied to transparent and nontransparent particle containers and flow lines of complex geometry which can be composed from the presented basic flow stream configurations.